These RNAs are of partly positive-sense and partly negative-sense polarity. The protein shell in contact or directly surrounding the viral nucleic acid (genome). These are morphological units that form capsids. Capsomeres consist of oligomers of one or more viral proteins. Cytopathic effect; could be due to apoptosis, necrosis, or syncytium formation. Full-length plus-strand template RNA complementary to the minus-strand genomic RNA. Cytotoxic T lymphocytes. Defective interfering viruses. DI virus particles contain a smaller viral genome, are noninfectious, and need the help of infectious (wild-type) virus for replication but, in turn, interfere with replication of homologous infectious (standard) viruses. The portion of the transmembrane protein that remains exposed outside of the cell or virus particle. These are the The process of internalization of external macromolecules or viruses, which involves specific binding to cell surface receptors. Viruses use this mechanism to enter into host cells. In this process, clathrin-coated vesicles and subcellular organelles like endosomes and lysosomes are involved. The viral membrane containing the lipid bilayer and associated proteins, which surround the nucleocapsid of enveloped viruses and form the outermost barrier of the enveloped virus particle. Viruses that possess an envelope or membrane surrounding the nucleocapsid. For enveloped viruses, the naked nucleocapsid is not infectious. The state of existence of nucleic acid molecules that do not became integrated into host cell chromosomes. They exist and multiply independently within the cell nucleus or cell cytoplasm. Virus mutants that are not neutralized by antibodies. These viruses possess amino acid change in the epitope and therefore no longer bind to the neutralizing antibody. The mechanism for transporting intracellular transmembrane or secretory proteins from intracellular compartments to the cell surface or extracellular environment. In this process, various subcellular compartments, like endoplasmic reticulum (ER) and Golgi complexes, are involved in protein transport. The complete genetic information (DNA or RNA) of an organism. In this process, one or several carbohydrate groups are attached to proteins during their transport through the exocytic pathways. Sugar residues are attached at specific sites to amino acids such as serine or threonine (for o-linked) or asparagine (for N-linked) carbohydrate moieties. These carbohydrate moieties are also called glycans. Glycosphingolipid.
Hepatitis B virus. These structures are spiral, springlike, and flexible rods. The RNA genome in a helical capsid is either exposed (influenza viruses) or enclosed (paramyxoviruses, rhabdovirus) by the nucleoprotein molecules constituting the nucleocapsid. Subtype specificity of influenza type A viruses. H denotes hemagglutinin (H1–H14), and N stands for neuraminidase (N1–N9) subtypes. Human immunodeficiency virus. Herpes simplex virus. Human rhinovirus strain 14. Intracellular adhesion molecule-1, the receptor for rhinoviruses. Icosahedron is a structure with a twofold, threefold, and fivefold rotational symmetry. It is a polyhedron with 20 faces, 12 vertices, and 30 edges. Most icosahedral viruses have 60 (multiple of 60) subunits (e.g., polioviruses, togaviruses). Microscopic structures, produced in some virus-infected cells consisting of viral proteins, nucleic acids, and cellular elements (particularly cytoskeletal elements). Inclusion bodies can be intranuclear (herpesviruses), intracytoplasmic (paramyxoviruses). Internal ribosome entry site. Lateral bodies found in poxviruses. Lymphocytic choriomeningitis virus, a member of the Arenavirus group. Large T antigen (ST = small T antigen) of SV40. Multiplicity of infection, that is, infectious units adsorbed per cell. These viruses do not have any membrane and the nucleocapsids represent the infectious virus. Nucleoprotein. Nuclear targeting (or localizing) signal. The complete nucleic acid-protein complex of a virus particle. Sometimes the term viral ribonucleoprotein (vRNP) is used to indicate nucleocapsid (e.g., vRNP of influenza viruses). Open reading frame. A circular nucleic acid structure of single-stranded (ss) DNA or RNA with a double-stranded stem at the end produced by intrastrand hybridization due to partial complementarity of the nucleic acid sequences at both the 5′ and 3′ termini of ssRNA or DNA. The panhandle structures function as the promoter and are important for transcription and replication. Plaque-forming unit. Uptake of particles by cells not totally dependent on receptor-mediated endocytosis. The particle on the surface is engulfed by the cell membrane into a phagocytic vesicle. These phagocytic vesicles then undergo similar changes as the endosome. Poxviruses enter cells by phagocytosis. Polyadenylation at the 3′ end of an RNA molecule. The term often used to indicate a structural unit containing one or more nonidentical protein subunits. Promoters are used as a building-block for virus capsid assembly.
RNA-dependent RNA polymerase, also called RNA transcriptase and RNA replicase. The RNA strand of the same polarity as the mRNA-encoding proteins is called positive-, plus-, or + strand RNA. When the RNA is of polarity opposite to the mRNA (i.e., cannot code for a protein), it is called negative-, minus-, or – strand RNA. Ribonucleoprotein. Respiratory syncytial virus. Reverse transcriptase, RNA-dependent DNA polymerase. Small T antigen (LT = large T antigen) of SV40. Structural proteins are those proteins that are found in virions either as components of capsid or envelope. Nonstructural proteins are those virally encoded proteins produced in the infected cells but not found in virions. Nonstructural proteins are usually catalytic and regulatory in nature and are also involved in modifying host functions. When all cells in the culture are infected simultaneously. Cells are infected at a high MOI (>5) and at low temperatures (4°C). Then the temperature is raised to 37°C to permit entry and uncoating of all cell-bound viruses at the same time. Cells possessing multiple nuclei are formed due to fusion among a number of cells. Usually viruses that can undergo fusion at a neutral pH (paramyxoviruses, retroviruses) produce syncytium. A mutant virus that will replicate at a permissive (low) temperature but not at the nonpermissive or restrictive (high) temperature. This phenotype is usually caused by missense mutations of one or more nucleotides, causing alteration of amino acid(s) of a protein that cannot assume the functional configuration at the nonpermissive (restrictive) temperature. Trans-Golgi network. These are membrane proteins that are anchored to the membrane by spanning the lipid bilayer of the membrane via transmembrane domains. These proteins can be classified as type I (e.g., influenza virus HA), type II (e.g., influenza virus NA), type III (e.g., influenza virus M2), or complex (e.g., coronaviral El) depending on the orientation of the NH2 and COOH termini (type I, II, or III), cleavage of signal peptide (type I), and multiple transmembrane spanning domains (complex). The entire virus particle. It usually refers to infectious or complete virus particle as opposed to noninfectious or defective virus particles. Vesicular stomatitis virus. A neurotropic variant of WS/33 (H1N1), a human influenza virus isolated in 1933 (
Viruses are unique life forms different from all other living organisms, either eukaryotes or prokaryotes, for three fundamental reasons: (1) the nature of environment in which they grow and multiply, (2) the nature of their genome, and (3) the mode of their multiplication. First, they can function and multiply only inside another living organism, which may be either a prokaryotic or eukaryotic cell depending on the virus. Viruses are acellular and metabolically inert outside the host cell and are obligatory parasites. Although there are other examples of obligatory parasites among the eukaryotes and prokaryotes, the nature of the intimate relationship between viruses and their host (i.e., environment) is much different. For example, some viruses extend their parasitic behavior to another level of mutual coexistence with their host, that is, they not only exist intracellularly but can, and do in some cases, integrate their genome into the genome of their host and thus tie their fate to the fate of the host. In fact, under these conditions, the integrated viral genome behaves like a host gene(s), undergoing similar regulatory control in transcription and replication and similar evolutionary changes as do the host gene(s). Second, whereas all other living forms can use only DNA (and not RNA) as their genetic material (genome) for information transmission from parent to progeny, viruses can use either DNA or RNA as their genome, that is, some viruses can use only RNA (and not DNA) as their genetic material. Therefore, these classes of RNA viruses have developed new sets of enzymes for replicating and transcribing RNA from an RNA template, as such enzymes (RNA-dependent RNA polymerase or RDRP) are not normally found in either eukaryotic or prokaryotic cells. Finally, all eukaryotic and prokaryotic cells divide and multiply as a whole unit, that is, 1 → 2→ 4 → 8 and so on. However, viruses do not multiply as a unit. In fact, they have developed a much more efficient way to multiply just as complex machines are made in a modern factory. Different viral components are made separately from independent templates, and then these components are assembled into the whole and infectious units, also called virus particles (virions), just as the complex machines are efficiently assembled from individual components. In this chapter, I will discuss aspects of viral morphology, the mode of viral replication, and viral morphogenesis.
Viruses are a heterogenous group of microorganisms that vary with respect to size, morphology, and chemical composition. The size of virions ranges from 20 nm (parvovirus) to ∼300 nm (poxvirus) in diameter, as compared to the size of Properties of the Virions of the Major Genera of DNA and RNA Animal Viruses Genome, the number indicates the segments of RNA present in the virus particle. All RNA genome is haploid except retrovirus (diploid). D = double-stranded; S = single-stranded. Ambisense (contains coding for protein on both genomic and complementary RNA strands). Circular helical nucleocapsid. Insect iridoviruses have no envelope; vertebrate members are enveloped. Reverse transcriptase (RT). Diploid, two molecules of the same RNA (+ strand) segment are present in one virus particle. Pleomorphic including filamentous forms. The capsid structure of mature retroviruses is not fully known, although it appears icosahedral. Length × width × thickness. Some virus particles contain (+)-strand and others contain (–)-strand DNAs.Viruses Genome nature Envelope Shape Genome polarity Size (nm) Transcriptase in virion Symmetry of nucleocapsid Enterovirus – Icosahedral + –20–30 – Icosahedral Rhinovirus S, – Icosahedral + 20–30 – Icosahedral Calicivirus S, 1 – Icosahedral + 20–30 – Icosahedral Alphavirus S, 1 + Spheroidal + 50–60 – Icosahedral Ravivirus S, 1 + Spheroidal + 40–50 – Icosahedral Orthomyxovirus S, 8 + Spheroidal – 80–120 – Helical Paramyxovirus S, 1 + Spheroidal – 100–150 + Helical Coronavirus S, 1 + Spheroidal + 80–220 – Helical Arenavirus S, 2 + Spheroidal ± 85–120 – Helical Bunyaviras S, 3 + Spheroidal ± 90–100 + Helical Retrovirus S, 1 + Spheroidal + 100–120 + Icosahedral Rhabdovirus S, 1 + Bullet-shaped – 175×70 + Helical Reovirus D, 10 – Icosahedral ± 70–80 + Icosahedral Orbivirus D, 10 – Icosahedral ± 50–60 + Icosahedral Filovirus S, 1 – filamentous – ≥80 × 800 + Helical Papillomavirus D, circular – Icosahedral ± 55 – Icosahedral Polyomavirus D, circular – Icosahedral ± 45 – Icosahedral Adenovirus D, linear – Icosahedral ± 70–80 – Icosahedral Herpesvirus D, linear + Spheroidal ± 150 – Icosahedral Iridovirus D, linear + Spheroidal ± 125 × 300 + Icosahedral Poxvirus D, linear + Brick-shaped ± 300× 240 × 140 + Complex Parvovirus S, linear – Icosahedral +– 20 – Icosahedral
The chemical composition of a virus depends on the nature of that virus, that is, the nature of the viral genome (RNA or DNA), the composition of the protein shell called the viral “nucleocapsid” surrounding the genome, and the presence or absence of viral membrane depending on whether the virus is enveloped or naked. All viruses have nucleocapsids and therefore contain nucleic acids and proteins. The nucleic acid is the genome that contains the information necessary for viral function and multiplication, and this information is passed from the parent to progeny viruses. Some viruses contain extragenomic nucleic acid, for example, tRNA in retroviruses and ribosomal RNA in arenaviruses. Viral proteins have three primary functions. They (1) provide the shell to protect the nucleic acid from degradation by environmental nucleases, (2) facilitate transfer of the genome from one host to another, and (3) provide many of the enzymatic and regulatory functions needed for transcription and replication so that viruses can survive, multiply, and perpetuate. In addition to the capsid shell, many viruses also possess an envelope (or viral membrane) around the nucleocapsid. The envelope in these viruses is critical for transmission of those viruses from one host to another host. The naked nucleocapsids of enveloped viruses are noninfectious because they lack the receptor binding protein. The viral envelope contains lipids and carbohydrates in addition to “envelope- or membrane-associated” viral proteins. The viral genome codes for most, if not all, of the proteins associated with the viral envelope. Lipids of the viral membrane are synthesized by the host cell and derived from the host cell. Therefore, viral lipid composition varies depending on the host cell in which the virus grows and also on the type of the cellular membrane (e.g., ER, Golgi, plasma or nuclear membrane) from which the particular type of virus buds. The carbohydrate content of the viral envelope is usually determined by the nature of glycosylation (N-glycosylation, O-glycosylation, complex versus simple sugar addition) of the viral envelope proteins, which may in turn undergo other modifications such as myristylation, palmitoylation, sulfation, and phosphorylation.
Genomes of different viruses are widely diverse in size and complexity. Some are comprised of DNA, others of RNA. As mentioned earlier, only in viruses is RNA known to function as a genome. Viral DNA genomes vary in complexity ranging from 5 kb containing 5–6 genes (parvoviruses, SV40) to 300 kb (avipoxviruses) containing more than 200 genes. Some DNA genomes are double-stranded (SV40), some are partially double-stranded (hepatitis B virus), and still others are single-stranded (parvoviruses) ( Replication of DNA Viruses ds = double-stranded; ss = single-stranded. Virions contain DNA-dependent RNA transcriptase and many other enzymes, but not DNA-dependent DNA polymerase.Virus Form of DNA Polymerase Activity Presence in virion Replication site in cell Papovaviruses ds Host DNA – Nucleus Adenoviruses ds Viral DNA – Nucleus Herpesviruses ds Viral DNA – Nucleus Poxviruses ds Viral DNA – Cytoplasm Parvoviruses ss Host DNA – Nucleus Hepadnaviruses Partially ds Viral Reverse transcriptase (RT) + Nucleus/cytoplasm
RNA genomes of viruses also vary in complexity but not as widely as do DNA genomes. They range from ∼7 kb (rhinoviruses) to ∼30 kb (coronaviruses). Coronaviral RNA represents the largest stable single-stranded RNA found in nature. Viral RNA can be single- or double-stranded ( Replication of RNA Viruses ss = single-stranded. ds = double-stranded; + or – indicates positive or negative polarity. ± = ambisense genome.Virus Form of RNA Source of nucleic polymerase Nature of polymerase activity Presence of polymerase in virion Viral replication site within host cell A Paramyxovirus, Rhabdovirus ss Viral RNA-dependent RNA polymerase (RDRP) Cytoplasm B. Bunyavirus, Arenavirus ss Viral RDRP Cytoplasm C. Orthomyxovirus (Influenza virus) ss (–), segmented Viral RDRP Nucleus D. Rotavirus, Reovirus, Orbivirus ds Viral RDRP Cytoplasm E. Picornavirus (Poliovirus, Hepatitis A), Togavirus (Sindbis virus), Coronavirus ss (+), unsegmented Viral RDRP – Cytoplasm F. Retrovirus, (HIV) ss (+), unsegmented, diploid Viral Reverse transcriptase Nucleus
The viral RNA genome may be unsegmented, consisting of a single RNA molecule, or segmented, consisting of multiple segments. Usually, viral genomes are haploid, but some are diploid (e.g., retroviruses; Features of the retrovirus genome. The diploid RNA genome includes the following from 5′ to 3′: the m7Gppp capping group, the primer tRNA, the coding regions, M6A residues (m), and the 3′ poly(AAAAA) sequence.
Organization of genes in the RNA genome varies between different groups of viruses. For positive-strand naked RNA viruses (e.g., polioviruses), which are translated into a single large polyprotein, the 5′ end of the genome is not capped but is rather covalently linked to a small protein, VPg ( Organization of picornaviral genome (RNA) and its translation products. P1, P2, and P3 indicate three intermediate precursor proteins cleaved from the polyprotein. These precursor proteins are further cleaved by virus-encoded proteases into mature functional proteins. Numbers in parentheses indicate molecular weights in thousands. hr and gr indicate host range and guanidine resistance determinants, respectively. 2A and 3C are proteinases involved in cleavage of the polyprotein and precursor proteins into mature viral proteins. Vpg, VP0, etc. indicate specific viral proteins.
For unsegmented minus-strand RNA genomes, the order of genes for both rhabdo- and paramyxoviruses are similar. Structural genes for capsid (N and P proteins) and envelope proteins are at the 3′ half, and the large polymerase (L) gene occupies the entire 5′ half of the minus-strand RNA genome ( Genome of unsegmented negative-strand RNA viruses (vesicular stomatitis virus [VSV] and Sendai viruses). Numbers underneath rectangles represent the number of nucleotides in each gene (shown above the line).
Proteins are major constituents of the viral structure, and their main functions, as indicated earlier, are to protect the nucleic acid from nucleases and to provide receptor-binding site(s) for virus attachment, which is required for efficient transmission of virus from one host to another. Viral proteins can be classified as either nonstructural or structural. Nonstructural proteins are those encoded by the virion genome, and expressed inside the virus-infected host cells, but not found in the virion particles. These nonstructural proteins usually have regulatory or catalytic functions, which are involved either in viral replication or transcription processes or are involved in modifying host functions. Structural proteins are broadly defined as those proteins found in virus particles. The majority of these structural proteins constitute the viral capsid or core, and are intimately associated with the viral genome to form the nucleocapsid. The cores of some viruses also contain regulatory or catalytic proteins as minor structural proteins (e.g., proteins with enzymatic functions, such as transcriptase (RDRP) or reverse transcriptase (RT) (
In addition to having viral capsids, the enveloped viruses possess membranes (or envelopes) surrounding the viral capsids. These viral membranes, as noted earlier, contain lipids derived from the host membrane and proteins specified by the viral genome. Two types of proteins are found in the viral membrane: transmembrane proteins and matrix proteins.
Transmembrane proteins, which can be either type I (such as influenza virus hemagglutinin), type II (such as influenza virus neuraminidase), and type III (such as influenza virus M2) depending on their molecular orientation; or complex proteins, containing multiple transmembrane domains (such as E1 glycoprotein of coronaviruses). Enveloped viruses may contain either only one (as in the G protein of VSV), two (as in the HN and F proteins in paramyxoviruses) or multiple transmembrane proteins (as in the influenza viruses, herpesviruses, poxviruses, etc.) on their envelope. These transmembrane proteins are often glycosylated via N- or O-glycosidic bonds, and their carbohydrate moieties can be comprised of simple sugars, usually consisting of mannose molecules, or complex sugars, including galactose, glucosamine, galactosamine, fucose, and mannose, as well as also sialic acid residues. Proper glycosylation of viral proteins is often important to provide for the necessary molecular stability, solubility, oligomer formation, and intracellular transport of viral proteins, as well as for modulating the host immune response, including epitope masking and unmasking. These glycans also may be important in apical sorting of proteins within polarized epithelial cells. It often is the case that one or more of these transmembrane proteins are involved in providing important functions in the processes of receptor binding, fusing of the viral envelope, uncoating of the viral genome, releasing of mature viruses from the infected cells, and spreading of viruses from cell to cell (e.g., function of NA, the neuraminidase protein, in releasing influenza viruses). These envelope proteins are also important for host defense, where they elicit neutralizing antibodies as well as CTL response against the virus infection in infected hosts and therefore play a critical role in vaccination and protection against viral infections.
In addition to the transmembrane proteins, the majority of these enveloped viruses also contain another type of membrane protein called a
Viruses vary greatly in size and shape. They can be either spherical or cylindrical (rod-shaped) ( Schematic presentation of different forms of viral structures. C = capsid; S = spike on viral envelope; E = viral lipid envelope; NC = nucleocapsid (i.e., capsid proteins in association with either RNA or DNA); M1 = matrix protein of influenza virus; LB = lateral bodies present in poxviruses; ss = single-stranded, ds = double-stranded RNA or DNA. Transmission electron micrographs of influenza virus ( Scanning electron micrographs of influenza viruses budding from infected cells. Spherical (
The formation of the viral capsid and its shape is primarily determined by the three-dimensional structure of the capsid proteins, which in turn is determined by the specific amino-acid sequence encoded by the viral nucleic acid. The amino-acid sequence is considered the primary structure of the protein, whose three-dimensional structure is composed of secondary structures such as α helices, β sheets, and random coils. These secondary structures interact with each other, forming the tertiary and quaternary structures, which are usually stabilized by noncovalent interactions (sometimes by covalent disulfide linkages), and represent folding of the proteins into relatively stable structures of microdomains (e.g., globular heads). In addition, extended and flexible regions of the proteins, called hinges, are also present, and these hinges become important for interaction with other members of the protein subunits that form the capsomeres. In most viruses, contacts between capsomeres are repeated, exhibiting a symmetry. This is a process of self-assembly driven by the stability of interaction among the protein subunits forming the capsomeres and the capsomeres forming the capsid. Viral capsids have either a helical (springlike) or icosahedral-based (cuboidal or spherical) symmetry.
Helical capsids are usually flexible and rodlike. The length of the helical capsid is usually determined by the length of the nucleic acids, that is, some defective interfering (DI) viruses having shorter nucleic acids will have a shorter helical nucleocapsid (e.g., DI RNA of VSV). Helical capsids can be naked, that is, without an envelope (e.g., tobacco mosaic virus). However, there is no known example of an animal virus with a naked helical nucleocapsid. All animal viruses with helical capsids found to date are enveloped. However, such helical capsids when enclosed in an envelope can appear to be either rod-shaped (e.g., rhabdoviruses) or spherical (e.g., orthomyxo- or paramyxoviruses), indicating that the helical capsid in these viruses is flexible (
Viruses with icosahedral capsids possess a closed shell enclosing the nucleic acid inside ( Structure of representative RNA and DNA viruses as determined by cryoelectron microscopy: (
To survive, viruses must multiply. Since viruses cannot multiply outside the host cell, they must infect host cells and use cellular machinery and energy supplies to replicate and produce the progeny viruses, which must in turn infect other hosts, and the cycle continues. Host–virus interaction at the cellular level is therefore obligatory for virus replication. Specific host cells can be either nonsusceptible (i.e., resistant or nonpermissive) or susceptible (i.e., permissive) to a particular virus. Nonsusceptibility of cells can be either at the attachment and entry phase (e.g., lack of a suitable receptor for a virus at the cell surface), at the intracellular phase (i.e., a block in synthesis of viral macromolecules), or at the assembly and exit phase. Following infection, viruses can cause abortive (nonproductive) or productive infection. Only productive infection yields infectious progeny virus particles. Following either abortive or productive infection, the host cell may survive or die (i.e., the cytopathic effect [CPE]). CPE caused by a virus does not necessarily indicate the permissiveness of a cell to a virus leading to productive infection. The viral genome in abortive infection may be degraded or may integrate into the host DNA or exist as extrachromosomal (episomal) DNA in the surviving cell. The growth properties of such cells may be altered, including the possibility that they may become transformed and cancerous. Alternatively, cells containing the integrated viral DNA may behave normally, exhibiting little change in their normal properties. Malignant transformation of the infected cells often depends on the site of viral genomic integration leading to activation of cellular oncogenes, disruption or inhibition of tumor suppressor genes, or synthesis of the viral oncogene products that are encoded by the virus in its genome. In the infected cells, the virus genome may remain dormant, resulting in a latent infection, and it can be activated later, producing infectious viruses, as occurs with herpesviruses. Alternatively, infected cells may yield virus at a low level without affecting cell survival, resulting in persistent infection, as occurs with LCMV.
The effect of virus infection has been studied at both the cellular and organismic levels. At the organismic level, it is called “viral pathogenesis,” while at the cellular level it is called the “cytopathic effect” (CPE). Under these conditions, cells may undergo morphological changes, including rounding, detachment, cell death and cell lysis (either apoptotic or necrotic), and syncytium (giant multinucleated cell) formation as well as inclusion body formation. Many of these changes are caused by the toxic effects of viral proteins affecting host macromolecular synthesis, including DNA replication, DNA fragmentation, mRNA transcription, translation, protein modification, and degradation, as well as other cellular synthetic and catalytic processes. Furthermore, since the same cellular machineries are directed toward viral macromolecular synthesis, the host is deprived of their functions. In addition to direct cell killing, virus infection can indirectly cause injury to tissues in a complex organism, as a result of complex host-viral immune interactions (i.e., immunopathology), as well as by cytokine production causing inflammatory reactions.
It is evident from the foregoing that, for successful replication of a virus, it must find susceptible host cells, and it must be able to attach itself to and penetrate into the host cell, and be uncoated, rendering the viral genome available for interaction of the viral and cellular machineries for transcription, translation, and replication of the viral genome. Finally, the newly synthesized viral components must be assembled into progeny viruses and released into the medium (outside environment) to infect other hosts. Whether with the cultured cells in laboratory or the complex organisms in nature, the virus-host interaction always occurs at the level of single cells. Thus, the viral infectious cycle (also known as the viral growth cycle, or replication cycle) can be divided into different phases, namely: (1) adsorption (attachment), penetration, and uncoating; (2) transcription, translation, and replication; and (3) assembly and release.
Viral adsorption is defined as the specific binding of a virus to a cellular (host) receptor. It is a receptor to ligand interaction in which viruses function as specific ligands and bind to the receptors present on the cell surface. Ligand functions of the virus are provided by the specific viral proteins present on the surface of the virus. For naked (i.e., non-enveloped) viruses, this function is performed by one of the capsid proteins, and for enveloped viruses, one of the membrane proteins functions as the ligand (variously also known as the receptor-binding protein, viral attachment protein or antireceptor) for the host receptor. Usually only one viral protein provides the receptor binding function, although one or more cellular proteins can function as receptor and coreceptor. For enveloped viruses, a classic example of a viral ligand (i.e., receptor-binding protein) is influenza virus hemagglutinin (HA), and its receptor-binding site is present on the globular head of the HA spike. For non-enveloped viruses, a classic example of a viral ligand is the VP1 of rhinoviruses. When five VP1 proteins are packed together within the viral capsid structure, the confluence of these grooves forms a depression called a canyon. The canyon has been shown to be the site for interaction between human rhinovirus-14 (HRV-14) and the cellular molecule ICAM-1 (receptor for rhinovirus). The amino acids lining the floor of these canyons are highly conserved, but residues on the surface of the canyon are variable ( Key features in the function of cellular receptor interactions with an invading virus, like a typical picornavirus. (
The cellular receptors of many viruses have been recently identified. Cellular receptors must be present on the cell surface and are either carbohydrates, lipids, or proteins. Sialooligosaccharides present on either glycoproteins or glycolipids function as receptors for orthomyxoviruses, paramyxoviruses, or polyomaviruses; phosphatidylserine and phosphatidylinositol are the likely receptors for VSV; and immunoglobulin superfamily molecules (CD4 for HIV, ICAM-1 for rhinovirus) as well as hormone or neurotransmitter receptors function as receptors for a number of other viruses (e.g., epidermal growth factor for vaccinia viruses, β-adrenergic receptor for reovirus, acetylcholine receptor for rabies virus). Some viruses have more than one receptor, one being the primary receptor and the other a coreceptor. A classic example of this is the case of CD4 and chemokine receptors (CXCR4, CCR5, etc.) respectively functioning as the receptor and coreceptor for HIV. Both the receptor and coreceptor are needed for productive HIV infection, although only one viral protein (gp 120) provides the receptor-binding sites for both receptor and coreceptors. Receptor–virus interaction is a major reason for the host and tissue tropism of viruses. Recent studies have shown that lack of a specific coreceptor on a cell's surface provides resistance to HIV infection in some persons. Receptor-virus interactions are specific, and the noncovalent binding is independent of energy or temperature. Thus, the kinetics of viral binding to cells can be determined at 4°C, which serves as a research aid since their interaction at that temperature prevents viral penetration and uncoating. Therefore, binding virus to cells at 4°C and subsequently raising the temperature to 37°C can be used to infect cells synchronously and to study the subsequent events such as uncoating and penetration of virus into host cells. The time course of viral adsorption follows first-order kinetics and is dependent on virus-to-cell concentration. Usually, cells contain a large number of receptors: in the range of 104–105 per cell.
Following specific ligand-to-receptor interaction, the next steps in virus replication include penetration of virus into the host cell and uncoating of the viral genome, which are energy-dependent processes and can be prevented experimentally in the laboratory by subjecting the virus-cell complex to low temperatures (4°C). Penetration refers to entry of the surface-bound virus particles inside the cell, where they either exist free in the cytoplasm or inside the host cell vesicles (usually within endosomes). Quantitatively, penetration of virus particles is measured by the loss of the ability of antiviral antibodies to neutralize the cell-bound virus particles after adsorption, an effect that occurs because, after the viral particles have entered the cell, they are protected and no longer accessible to antibodies outside the cell. Uncoating, on the other hand, refers to disruption of virus particles, causing partial or complete separation of nucleic acid from the capsid, and is needed for initiation of transcription and translation of the viral genome. Uncoating can be assessed by, among other things, alterations in viral morphology or viral density, release of nucleocapsid and membrane proteins from enveloped virus particles, as well as by the accessibility of the viral genome to nucleases. For such viruses as orthomyxovirus and poliovirus these processes are separated temporally (i.e., penetration is followed by uncoating in the cytoplasm), but for others both penetration and uncoating occur simultaneously at the cell surface (e.g., paramyxoviruses, HIV). Uncoating refers to the step in which the viral genome becomes functional either transcriptionally or translationally. However, complete separation of nucleic acid from all capsid proteins is not required for most viruses. For naked viruses, uncoating is a postpenetration process that occurs either in the endosome or in the nucleus. Viruses that undergo uncoating in the cytoplasm following endocytosis require low pH (∼5) in the endosome for uncoating, whereas viruses that undergo fusion at the cell surface can undergo uncoating in a pH-independent manner.
Naked viruses like the RNA-based picornaviruses enter into the cytoplasm of the infected cells via receptor-mediated endocytosis ( Receptor-mediated endocytosis of viruses like polioviruses (steps 1 through 4a,b). The virus binds to
For enveloped viruses, uncoating occurs through fusion of the viral membrane with the cellular membrane using either pH-independent or pH–dependent pathways. In the pH independent pathway, virus penetration and uncoating occur simultaneously and at the cell surface after virus–host interaction. This is best illustrated by the entry process of paramyxoviruses and retroviruses (e.g., HIV). In both cases, viruses bind to the cell surface receptors (i.e., sialic acid present either on the cell surface glycolipids or glycoproteins for paramyxoviruses and the receptor protein CD4 and coreceptors for HIV). Either one (gp160 for HIV) or two (F and HN for paramyxovirus) separate viral glycoproteins are involved in this binding and fusion processes. One of these proteins must be cleaved in the infecting virus for fusion to occur (examples being gp160 → gp120 and gp 41 for HIV and F→F1 and F2 for Sendai virus). For HIV, the gp120/gp41 complex undergoes conformational changes after binding to the cellular receptor and coreceptor, releasing the hydrophobic domain of gp41, which then functions as a fusion peptide and causes fusion of the viral membrane with the plasma membrane, thereby releasing the nucleocapsid containing the viral RNA and reverse transcriptase into the cytoplasm. Subsequently, cyclophilin A, present in HIV particles, aids in the uncoating process by destabilizing the capsid and initiating reverse transcription of the viral RNA. For paramyxoviruses, HN protein binds to the sialic acid on the cell surface receptor and induces, in some way, conformational changes in the other viral envelope protein, known as the F1/F2 complex, and thereby facilitates the fusion domain of F1 to cause fusion between the viral membrane and the plasma membrane, and release of the viral nucleocapsid containing the transcriptase (RDRP) into the cytoplasm. For paramyxovirus, the entire viral replication process takes place in the cytoplasm, whereas for retroviruses the proviral DNA is formed in the cytoplasm after reverse transcription of the viral RNA and is then transported into the nucleus for integration and transcription. How the receptor-protein interaction facilitates conformational changes leading to fusion of the viral and cellular membranes in a pH independent manner is not fully understood. Furthermore, fusion for these viruses occurs not only between viruses and host cells but also between virus-infected cells expressing the cleaved viral membrane proteins on the cell surface and uninfected cells containing the receptors (and coreceptors) present on the cell surface. These cell-to-cell interactions lead to formation of syncytium or multinucleated giant cells. Such multinucleated giant cells are important diagnostic markers for a number of viral infections (e.g., respiratory syncytial virus [RSV], mumps, measles viruses). The process of fusion of HIV-infected cells to uninfected CD4+ T cells is implicated in AIDS pathogenesis, which causes depletion of CD4+ T cells in HIV infected persons.
For other enveloped viruses like VSV and influenza viruses, penetration and uncoating are two separate events. Following receptor binding, these other viruses enter the cytoplasm by receptor-mediated endocytosis, and fusion and uncoating occur within the endosome in a pH-dependent (low pH of ∼5) manner. The fusion and uncoating of these viruses can be blocked by agents like monensin, which increases endosomal pH. For VSV, the G protein binds to the receptor and becomes activated for fusion at low pH, even though it remains uncleaved. Although the VSV G protein contains a hydrophobic fusion region, the mechanism of its fusion process within the endosome is not well understood. The fusion and uncoating processes are best understood at the molecular level for influenza viruses. Again, for influenza viruses, although fusion and uncoating occur simultaneously, they are considered two separate events. Following binding to sialic acid on the cell surface receptor, influenza virus undergoes receptor-mediated endocytosis and the cleaved HA trimer (i.e., HA1/HA2 heterotrimer complex) present on the viral membrane undergoes conformational changes at the low pH of endosomes (∼5).
Acidic pH specifically alters the structure of HA2, which attains the fusiogenic state. In conjunction with this process, HA1 becomes dissociated from the stem of the HA spike, and the fusion peptide present at the NH2 terminus of HA2, which normally remains buried in the protein interior of the HA trimer, is released and the polypeptide structural loop becomes transformed into a helix to form an extended coiled coil structure that relocates the hydrophobic fusion peptide toward and into the target (endosomal) membrane ( A model for the fusiogenic state of HA of influenza virus. HA in its native state is a trimer. However, for demonstration of the conformational changes during fusion, only a monomer is depicted in this figure. Panel Membrane fusion via a hemifusion intermediate. ( Schematic presentation of the infectious cycle of an influenza virus. The steps in the replication cycle are noted as 1 (attachment) through 12 (release). PB1, PB2, PA, NS1, NS2, NP, M1, HA, NA, and M2 are the virus-encoded proteins translated from 10 mRNAs, which are transcribed from eight vRNA segments of negative polarity.
Viral replication occurs either in the nucleus or in the cytoplasm of infected cells. For those viruses that replicate in the cytoplasm, which customarily are those with RNA genomes, except for the DNA-containing poxviruses, the uncoating process releases the viral nucleocapsid directly into the cytoplasm, which is the site of transcription and replication. For viruses that replicate within the nucleus, which tend to be the ones having DNA genomes with notable exceptions such as the RNA-containing influenza viruses and retroviruses, the viral nucleocapsids that are released in the cytoplasm after uncoating must be targeted into the nucleus. Nuclear targeting requires that these viral nucleocapsids contain proteins possessing nuclear targeting signal(s) (NTSs or NLSs), which are recognized by the cellular nuclear targeting machinery and translocated into the nucleus via nuclear pores. However, the stage of uncoating at which nuclear targeting takes place varies with viruses. For SV40, essentially the entire virus particle that has been taken into the cytoplasm is transported into the nucleus, and it is only in the nucleus that uncoating of the capsid occurs concomitant with release of the viral minichromosome. For adenoviruses, uncoating occurs at the nuclear pore where the viral nucleocapsid docks and the viral DNA is delivered into the nucleus through the nuclear pore. For influenza viruses, uncoating occurs during introduction of the nucleocapsid into the cytoplasm by dissociation of M1 from the vRNP. This M1-free vRNP is then transported into the nucleus. For retroviruses, not only uncoating but also additional biosynthetic processes — including reverse transcription of the RNA genome and synthesis of the double-stranded proviral DNA — occur in the cytoplasm. Then the retroviral DNA along with integrase is translocated into the nucleus for integration of the proviral DNA into the host genome. Transcription of the retroviral genomic and subgenomic mRNAs occurs only from the integrated proviral DNA in the nucleus. For hepatitis B virus, the partially double-stranded DNA, the viral genome following uncoating in the cytoplasm, becomes fully double-stranded and circularized in the cytoplasm, and then it is translocated into the nucleus for subsequent transcription of genomic and subgenomic mRNAs.
The “immediate events” in the viral replication cycle, those that occur following uncoating, vary with the nature of the viral genome. For plus-strand RNA viruses except retroviruses, translation of the viral RNA follows immediately after uncoating. The viral RNA extruded from the capsid is then used by the host translation machinery for directing protein synthesis (
From the transcription viewpoint, viruses can be classified into two major categories, that is, whether they possess a DNA genome or an RNA genome. Of the first group, the DNA genome of different viruses varies greatly in complexity between virus families, encoding from only 4 to 5 genes to more than 200 genes or open reading frames (ORFs). DNA viruses use DNA-dependent RNA polymerase, which can be either virus-specified (e.g., poxviral RNA polymerase) or host-specified (e.g., RNA
All DNA viruses except for the poxviruses transcribe and replicate their genomic material in the host cell nucleus. Poxviruses transcribe and replicate in the cytoplasm. In addition, all DNA viruses except poxviruses use host
An example of transcription of a small double-stranded viral DNA genome (SV40) is shown in Genome and transcription map of SV40 (top). The origin of replication is shown at the top of the inner circle. The numbers indicate the nucleotide position in the SV40 DNA, while zigzag markings indicate spliced introns. Different shaded regions indicate different protein-coding sequences. The bottom drawing shows the details of the transcription regulatory elements in the proximity of the “origin” region and the direction of the early and late transcription.
On entry into the cytoplasm of the infected cell, hepatitis B virus (HBV), a partially double-stranded DNA virus, uses virus-specified reverse transcriptase (P) to synthesize the complete circular DNA, which is then transported into the nucleus. Host cell Replication, transcription, and translation of hepatitis B virus (HBV) DNA. Four RNA classes: 3.5 kb (1), 2.4 kb (2), 2.1 kb (3), and 0.7 kb (4) are transcribed. The 3.5-kb product (#1) is used for full-length DNA (minus-strand) synthesis. Different classes of 3.5-kb product also function as mRNAs whose translation products are HBcAg, the polypeptide consisting of the PC-ORF (pre-core), and C-ORF (core) and P-ORF (P-protein, also called either polymerase or reverse transcriptase). The 2.4-kb mRNA (#2) makes a large protein consisting of the polypeptides PS1-ORF, PS2-ORF (presur-face), and S-ORF (surface protein). The 2.1-kb mRNA (#3) makes the S-ORF (surface) protein, and the 0.7-kb mRNA (#4) encodes the X-ORF protein.
Among the different families of RNA viruses, the RNA viral genome appears to be much less complex as compared to the genomes of the highly complex DNA viruses. However, these RNA viruses use multiple strategies to encode different mRNAs and different proteins. Unlike DNA viruses, the majority of the RNA viruses (except for retro-, orthomyxo-, and related viruses) replicate in the cytoplasm, so that their mRNAs cannot undergo RNA splicing. RNA viruses also possess genes for regulatory and catalytic proteins as well as for structural proteins. However, transcription of mRNAs encoding these proteins is not as strictly demarcated with respect to the timing of their genomic nucleic acid replication, as is found for DNA viruses. On the other hand, with RNA viruses there is a great deal of variation in the level of transcription of different viral mRNAs. The mRNAs of the major structural proteins — like the nucleoprotein (NP) and matrix (M) glycoproteins — are usually made in larger amounts as compared to the lower amount of mRNAs synthesized for catalytic (e.g., polymerases) proteins. For unsegmented negative-strand RNA viruses, the level of mRNA transcription is regulated by the promoter-proximal position of a gene (e.g., for VSV or paramyxoviruses, see For plus-strand icosahedral naked RNA viruses (e.g., poliovirus), the entire viral genomic RNA functions as the only mRNA and is translated from one ORF into a large polyprotein, which is then cleaved by specific proteases into different functional proteins representing the RNA polymerase and the capsid proteins (VP1, VP2, VP3, VP4), and so on ( For some enveloped plus-strand RNA viruses (e.g., togaviruses), the 5′ half of the viral genomic RNA encodes and is translated into nonstructural (catalytic) proteins involved in RNA transcription and RNA replication, whereas a separate subgenomic 26-S mRNA (+), made from an internal promoter on the minus-strand RNA template, encodes the structural proteins (i.e., capsid and envelope proteins). This 26-S mRNA is synthesized in a larger quantity than is the genomic length RNA. However, another group (flaviviruses) of enveloped plus-strand RNA viruses possesses one large ORF in its genomic RNA encoding a single large polyprotein, which, as is the case with picornaviruses, is cleaved into specific proteins by a virus-encoded proteinases. For coronaviruses, which contain a large plus-strand RNA genome of ∼30 kb, multiple subgenomic mRNAs are found. However, each of these mRNAs possesses the same 5′ leader (i.e., leader-primed transcription) and the common 3′ end containing poly(A) sequences. These mRNAs therefore contain the nucleotide sequence of more than one ORF. Usually, however, only the first ORF at the 5′ end of mRNA is translated into protein. Minus-strand RNA (-) viruses replicating in the cytoplasm may possess either one large genomic RNA molecule (unsegmented) or two or more different subgenomic RNAs (segmented). For those viruses that possess an unsegmented genomic RNA molecule (e.g., VSV), the viral genes are arranged sequentially in the genomic RNA (–) with stop, intergenic, and start (EIS) sequences ( Orthomyxoviruses, which are segmented, minus-strand RNA viruses, possess 8 RNA segments, which in total encode 10 mRNAs and 10 proteins for type A and B viruses. Orthomyxoviruses are transcribed and replicated in the nucleus. Orthomyxoviruses use a unique strategy to initiate transcription. They cannot initiate Transcription and replication of the influenza virus RNA (vRNA). ( Segmented ambisense RNA viruses (e.g., arenaviruses) on infection produce a subgenomic mRNA using the 3′ end of the genomic RNA as the template, and later on in the infectious cycle use the antigenomic RNA as the template to generate the mRNA with the same polarity as the 5′ end of genomic RNA. Viruses that possess double-stranded (ds) RNA viral genomes, such as reoviruses, are segmented and replicate in the cytoplasm. Their viral transcriptase, which is also present within the virus particles, synthesizes single monocistronic mRNAs from each dsRNA segment. Retroviruses, although possessing a plus-strand RNA genome, contain reverse transcriptase (RT) in the virion. Transcription of retroviral mRNAs occurs in the nucleus from the integrated proviral DNA template by the host RNA
Virions have evolved to become very efficient organisms that package a relatively small amount of genomic DNA or RNA in their capsids but use this information efficiently to generate the maximum number of functional proteins required to produce infectious progeny virions. For some viruses like VSV, all of the viral proteins encoded by the genome and produced in the infected cells including the transcriptase are incorporated into the virion and become structural components of virus particles. For these viruses, there are by definition no nonstructural proteins, that is, there are no proteins that are encoded in the virion genome and produced in the infected cells but not incorporated into the virion. However, for the majority of viruses, one or more nonstructural proteins, either catalytic (enzymatic) or regulatory, are synthesized in virus-infected cells. These nonstructural proteins are required for the infectious cycle but are not incorporated into virion particles. Both structural and nonstructural proteins are translated from viral mRNAs, and the majority of viral mRNAs (except in the case of picornaviruses) possess structural features similar to that of the host mRNA (i.e., they possess a cap at the 5′ end, a translation initiation triplet [AUG] in the context of Kozak's rule, as well as translation termination triplets and poly(A) sequences at the 3′ end). These viral mRNAs undergo cap-dependent ribosome binding and ribosome scanning to locate the proper initiation triplet, a process that does not provide any advantage over the host mRNAs during translation. Therefore, after infection, the virus must overcome two major problems to achieve successful replication: (1) viruses must somehow overcome competition from host mRNAs for using translation machineries, and (2) viruses that possess only a limited amount of coding information must still be able to generate the considerable number of functional proteins needed for replication. Viruses have developed a number of strategies to compete with host mRNAs for efficiently using the host translation machinery. These include the following. (a) Viral transcription machinery (especially in RNA viruses) are more efficient in generating high levels of mRNAs so that they can outcompete host mRNAs in translation. (b) Some viral proteins target and interfere with the host transcription machinery so that the host transcription level goes down or shuts off. Influenza viruses, however, use a novel system to their advantage. As mentioned earlier, one of the influenza polymerase proteins, PB2, recognizes, binds to, and cleaves the newly synthesized capped host hnRNAs around 13–15 nucleotides, and the capped oligonucleotide is used as the primer for mRNA synthesis. The cleavage of host hnRNAs, in turn, prevents host mRNA synthesis and processing. In addition, this virus interferes with nuclear export of the host mRNAs. (c) Some viruses modify the host translation machinery to use that machinery for its advantage while simultaneously shutting off host mRNA translation. This latter mechanistic approach is particularly evident for picornaviruses, which inactivate the cap-binding protein and modify the host translational factors (e.g., eIF2, eIF3/4B) and thus shut off cap-dependent host mRNA translation. However, picornaviral mRNA can still be translated efficiently because it does not have a cap at the 5′ end but rather possesses a unique RNA secondary structure known as an internal ribosome entry site (IRES) and is independent of Kozak's rule. The picornaviral mRNAs possessing an IRES can be translated efficiently in a cap-independent manner, while capped host mRNAs cannot be translated because of viral-mediated inactivation of some of the host translational factors. Viruses have developed different strategies to produce a relatively large number of functional proteins from a small amount of genetic information using both transcriptional (or posttranscriptional) as well as translational (or posttranslational) processing.
Transcriptional (or posttranscriptional) generation of different mRNAs. Double-stranded DNA viruses can use both of their DNA strands to transcribe mRNAs, thereby increasing potential transfer of information into proteins. Some viruses that make mRNAs in the nucleus (either RNA or DNA viruses) can generate different mRNAs from the same genomic strand by using either unspliced mRNA or electing alternative splicing sites, thus even causing frame shifts in the subsequent translation. Influenza viral proteins M1, M2, NS1, NS2, and SV40 proteins (such as VP1, VP2, and large T and small T antigens) are classic examples of generating different mRNAs and proteins through splicing. Some viruses use RNA editing (i.e., nontemplated nucleotide addition in the mRNA) to shift the translation frame. This latter technique is frequently used by paramyxoviruses to generate their V and C proteins. Hepatitis delta virus uses adenosine deaminase for RNA editing as part of the transcription process to generate its δ Ag-L antigen. Other viruses selectively use different promoters to generate genomic and subgenomic mRNAs (e.g., HBV, togaviruses). Translational (and posttranslational) generation of different viral proteins. The most common way to generate a number of functional proteins after translation is by proteolytic cleavage. These endoproteases, usually encoded by the virus, are sequence-specific and can generate a number of functional proteins from one large viral polypeptide. Classic examples of this type of cleavage activity are found with poliovirus (picornavirus) and flavivirus proteins. Poliovirus RNA is translated into a large polypeptide that sequentially undergoes endoproteolytic cleavage by different poliovirus proteases at specific amino-acid sites, generating 11 viral proteins (VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, VPg, 3C, 3D) and other intermediate proteins ( Different initiation codons are also used in bicistronic mRNAs to translate different proteins. Depending on the initiation codon used, either one or the other protein can be translated (e.g., NB protein and NA protein from the same mRNA in influenza virus type B). Usually, one of the initiation codons is favored, thus regulating the levels of the two proteins produced from one bicistronic messenger RNA. Another strategy, often used by retroviruses, is either translational frame-shift or translational suppression of termination codons. Translational frame-shift due to ribosomal slippage causes generation of the
The replication pathway of different viral genomes varies depending on the nature of the viral genome. The overall strategy of viral genome replication can be grouped into seven pathways depending on the nature of the genome ( Seven replication pathways of the DNA and RNA genome of viruses. Examples of different viruses with DNA or RNA genomes are indicated. ds = double-stranded; ss = single-stranded; + and – indicate positive and negative polarity.
Smaller DNA viruses (papova- and parvoviruses) rely on the host cell DNA polymerase, whereas more complex DNA viruses use their own virus-encoded DNA polymerase ( Replication pathways for viral DNA genomes. In panel
Hepatitis B virus DNA uses reverse transcription for replication (
Viral RNA genomes can be single-stranded and comprised either of a plus or minus strand, or double-stranded. Furthermore, while the genomes of some RNA viruses are segmented (multiple RNA molecules), others are nonsegmented (i.e., one RNA molecule) (
Plus-strand RNA viruses are copied into a complete minus-strand RNA, which then serves as a template for synthesis of more plus strands via replicative RNA intermediates (
Each segment of double-stranded viral RNA genome is replicated independently. First, the genome is transcribed to generate plus-strand mRNAs within the incoming virion core by the virion-associated RDRP. Next, the mRNA is used as a template by RDRP to synthesize the minus RNA strand, and thereby mRNAs become converted into double-stranded RNA, which is then packaged into progeny virion capsids.
Retroviruses contain a diploid genome consisting of two identical RNA molecules, a tRNA primer ( Reverse transcription of retroviral genomic RNA into double-stranded proviral DNA.
As indicated earlier, when compared to either eukaryotes or prokaryotes, viruses use a unique multiplication strategy to produce their progeny. All cells, either prokaryotic or eukaryotic, multiply as a whole unit from parent to progeny and in a geometric order, that is, from 1 to 2 to 4 to 8, and so duplicatively on. Viruses, on the other hand, do not multiply as units. Rather, they are assembled from component parts. Each component part of progeny virus particles is made separately, and they are often made in different amounts and at different locations and compartments within the host cell. These viral components are then put together to form the whole (infectious) virus particles (virions). In this assembly-line type of process, all individual viral components need not be assembled at the same time, and in fact, some components may be put together separately to form higher-ordered structures, that is, subviral particles (e.g., capsid), before they are assembled into a whole progeny virus particle. The number of steps involved and the complexity of the assembly process may vary greatly from one type virus to another. Some viruses, like the polioviruses, have only a few components to assemble, and yet others, like the pox or herpesviruses, have many components to assemble and their assembly compared to polioviruses is a far more complex process involving multiple steps.
With respect to the assembly processes, viruses can be classified into two major subclasses: naked viruses and enveloped viruses. Naked viruses consist of a nucleocapsid only (i.e., the capsid containing the genome and no envelope). The assembly of the protein capsid and incorporation of genomic nucleic acid into the capsid to create this nucleocapsid will render the virus particle infectious. For these viruses, the virus receptor-binding proteins are part of the capsid proteins. Enveloped viruses, however, are those in which the nucleocapsid is surrounded by a lipid membrane containing the transmembrane viral proteins. In enveloped viruses, one of the transmembrane viral proteins (and not the capsid protein) contains the receptor-binding protein.
The assembly of naked viruses occurs either in the cytoplasm (most RNA viruses) or nucleus (DNA viruses). For cytoplasmic viruses (e.g., the plus-sense RNA picornaviruses), the entire genomic RNA is translated into a single giant polyprotein (
For assembly of a naked virion to occur inside the nucleus, one of at least two distinct strategies can be used. The first of these would require that all capsid proteins, after their translation in the cytoplasm, must be transported into the nucleus either independently or cooperatively by forming a complex with other capsid proteins and that nucleocapsid assembly occurs around the viral genome in the host nucleus. This option is used by papovaviruses, whose DNA genomes, or minichromosomes, contain a single closed circular duplex DNA molecule complexed with cellular histone, which is organized into a nucleosome within the host nucleus. Papovaviral capsid assembly then proceeds in a stepwise fashion around the viral minichromosome. The capsid of SV40, which is a member of this virus group, contains 360 copies of its major viral protein (VP1) assembled into 72 pentamers plus 30 to 60 copies of internal proteins VP2 and VP3. VP2 contains the full VP3 sequence plus 100 extra amino acids at the NH2 terminus, which are critical for interacting with the SV40 minichromosome. The papovaviral capsid proteins and minichromosomes assemble first into 200-S structures called provirions, which then mature into infectious virions. During this maturation, H1 histone protein is removed from the viral minichromosome and degraded.
Adenoviruses use a second type of strategy in which the capsid shell is first formed by the assembly of viral capsid proteins. Viral DNA, including core proteins, is then inserted into the empty capsid shells to form infectious virions. Both of these nuclear DNA viruses as well as the cytoplasmic naked RNA viruses are primarily released to the extracellular environment by cell lysis.
The assembly of enveloped viruses is much more complex than that of naked viruses. It involves not only nucleocapsid formation but also envelopment of the nucleocapsid and budding of virions from different cellular organelles and membranes. Subsequently, the virus is released into the extracellular environment. The assembly and the budding site on the cellular membrane varies with different groups of viruses. Some viruses, like the poxviruses and rotaviruses, bud from the endoplasmic reticulum (ER), while others, like the bunyaviruses, bud from the Golgi complex, and still others bud from the nuclear membrane, like the herpesviruses. Still other viruses (e.g., orthomyxo-, paramyxo-, rhabdo-, and retroviruses) use the plasma membrane (apical or basolateral) as the budding site.
Since the assembly processes of orthomyxo- and paramyxoviruses have been well studied, the steps involved in morphogenesis of these viruses will be discussed below in some detail. For comparison, rhabdovirus and retrovirus assembly will also be included as needed. First, I will discuss the steps that are common to both orthomyxoviruses and paramyxoviruses and then later point out the differences between these two viruses in terms of assembly and morphogenesis. As noted earlier, orthomyxo- and paramyxoviruses are enveloped RNA viruses that contain single-stranded RNA genomes of negative (minus) polarity, and they are assembled into nucleocapsids that have helical symmetry (
For elucidating viral assembly and budding processes, the viral structure can be separated into three major subviral components, each of which must be brought to the assembly site for morphogenesis. These subviral components are: (a) the viral nucleocapsid (or viral ribonucleoprotein [vRNP]) containing the vRNA, NP (nucleoprotein), and transcriptase complex, which together form the inner core of virus particle; (b) the matrix protein, which forms an outer protein shell around the nucleocapsid and constitutes the bridge between the envelope and nucleocapsid; and (c) the envelope (or membrane), which forms the outermost barrier of these enveloped virus particles, containing the virally coded transmembrane proteins and host cell lipids. Each of these virus groups (namely, orthomyxo-, paramyxo-, rhabdo-, and retroviruses) buds from the plasma membrane of infected cells. However, while the orthomyxo- and paramyxoviruses bud from the apical plasma membrane of polarized epithelial cells, both
With respect to the processes involved in virus assembly, there are two major differences between orthomyxo- and paramyxoviruses: (a) Since the viral genome of orthomyxoviruses is segmented, multiple RNA segments (8 separate RNA segments for influenza types A and B, 7 RNA segments for influenza type C viruses) must be incorporated into infectious virions, whereas only one large RNA molecule is packaged in infectious paramyxovirus particles. (b) Since the transcription and replication of orthomyxoviral RNA and assembly of these viral nucleocapsids (vRNP) occur in the host nucleus, the viral nucleocapsids must be exported out of the nucleus into the cytoplasm for the final stages of viral assembly and for budding. In contrast, for paramyxoviruses, all of these steps, including assembly of viral nucleocapsids, take place in the cytoplasm.
Analysis of the steps involved in putting these subviral components together in an orderly fashion into an infectious virus particle is critical to an understanding of the assembly and budding processes. Two steps are obligatory for virus assembly and morphogenesis to occur. First, all of these viral components (or subviral particles) must be directed and brought to the assembly site, that is, the apical plasma membrane in polarized epithelial cells for assembly and budding of orthomyxo- and paramyxoviruses. Obviously, this step is the first obligatory requirement in virus assembly, since, if different viral components are misdirected to different locations or parts of the cell, virus assembly and morphogenesis cannot take place. Second, the viral components must interact with each other to form the proper virus structure during morphogenesis. It is possible that viral components may be directed to the assembly site but that defective interaction among these components will not yield infectious particles. However, although these two steps are obligatory, they alone may not be sufficient to form and release infectious virus particles. Therefore, virus components may be directed correctly to the assembly site and then interact with each other to form virus particles, yet infectious viruses may not be released into the medium. Such abortive virus morphogenesis in HeLa cells infected with influenza viruses has been observed where virus particles are formed on the plasma membrane but not released (
Among the viral components, the greatest amount of information is available about the transport, sorting, and targeting of viral transmembrane proteins to the assembly site (prospective budding site) in the plasma membrane. It has been observed that the transmembrane viral proteins in virus-infected cells also preferentially accumulate at the virus assembly site, that is, the orthomyxo- and paramyxoviral proteins like HA, NA, F, and HN accumulate on the apical plasma membrane, whereas rhabdoviral and retroviral transmembrane proteins like the VSV G and the HIV gp160 proteins are targeted to the basolateral plasma membrane in virus-infected polarized epithelial cells (see
Although the nature of the cellular machineries that target either viral or cellular proteins to their appropriate apical or basolateral plasma membranes are not fully understood, it is now accepted that these viral proteins are targeted directly to apical or basolateral surfaces and that for apical versus basolateral targeting protein sorting occurs during the process of vesicular transport from the trans-Golgi network (TGN) to the plasma membrane. It has also been shown that separate sorting machineries (vesicles) are involved in targeting to the apical and basolateral surfaces. It is therefore expected that viral proteins must possess determinants that selectively interact with either the apical or basolateral sorting machineries of the host cells. Those proteins that are directed to the basolateral membrane, including the VSV (a member of family Rhabdoviridae) G protein, possess basolateral sorting determinants in their cytoplasmic tail region. However, the nature and location of the sorting determinant(s) of apical proteins are more complex. For example, type I apical viral proteins like influenza virus hemagglutinin (HA) and type II transmembrane proteins, like influenza virus neuraminidase (NA), have been shown to possess at least two apical sorting signals that function independent of each other (see
In addition to the transmembrane glycoproteins, other viral proteins and subviral complexes such as matrix protein and nucleocapsid must also reach the assembly site. How these viral components are also directed to the assembly site is not yet fully understood. The possibility exists that the matrix (M1) protein may be directed to the assembly site by its interaction with the transmembrane viral proteins using a piggyback mechanism. Similarly, viral nucleocapsids (or ribonucleoprotein) may be transported to the assembly site either independently or on the back of the M1 protein. A number of studies have shown that matrix proteins as well as matrix protein/nucleocapsid complexes interact with the glycoproteins in transit to the plasma membrane (see
The viral matrix protein is a key component in virus assembly and morphogenesis. It is the most abundant protein in virus particles and is the rate-limiting component in particle formation since the virus particle is greatly reduced when matrix protein synthesis is defective or reduced. However, particles with reduced amounts of glycoproteins can be formed efficiently, although such viruses may be less infectious, or noninfectious. Freeze-fracture electron microscopy has shown that, during bud formation in virus-infected cells and in virus particles, the matrix protein is present as a sheet between the lipid bilayer and the viral nucleocapsid. The results of these and other studies imply that the matrix protein is likely to interact with both the lipid bilayer of the membrane and its associated viral glycoproteins on the outer side, and with the viral nucleocapsid on the inner side of the virus particles. In virus particles, the matrix protein remains bound strongly to the nucleocapsid under conditions where membrane glycoproteins can be dissociated using nonionic detergents. It is only after further treatment with either low-pH (influenza virus) or high-salt (paramyxovirus) buffer that matrix proteins can dissociate from the viral nucleocapsids. Therefore, we have strong evidence of interactions between the nucleocapsids and the matrix proteins. However, a detailed analysis of the exact nature of these associations in virus-infected cells at different stages of the assembly process has yet to be undertaken.
Likewise, the location of the matrix protein and glycoproteins in virus particles and in virus-infected cells predicts an interaction between the matrix protein and glycoproteins. However, the interaction of matrix proteins with membrane glycoproteins has been rather difficult to demonstrate. Studies using morphological as well as biochemical analyses have demonstrated that the Sendai virus (a member of family Paramyxoviridae) M (matrix) protein can bind independently to either Sendai virus membrane glycoproteins (F or HN), and that this interaction can take place on the plasma membrane as well as during exocytic transport of F and HN proteins through the Golgi complex. Biochemical and morphological studies have also shown that interaction of the Sendai virus M protein with viral glycoproteins can occur in the absence of nucleocapsid (or NP) protein (
Although the majority of host proteins present on the cell membranes are excluded during the budding process, two classes of host components (the lipids and cytoskeletal components) do appear to be involved in virus morphogenesis. Viral lipids are directly borrowed from the cellular lipids, and depend on the site of virus budding. For example, viruses budding from the apical or basolateral membrane will incorporate the lipids present on one specific side of the plasma membrane. It has also been specifically suggested that glycosphingolipids (GSLs) and cholesterol, which are present in the host cell apical membrane, may play a specific role in targeting viral transmembrane proteins to the apical membrane domains (budding sites). For example, both type I (influenza virus HA, Sendai virus F) and type II (influenza virus NA, Sendai virus HN) proteins associate with TX100 detergent-insoluble lipid rafts enriched in GSL and cholesterol during exocytic transport to the viral assembly sites on the plasma membrane.
Cytoskeletal components facilitate both the transport of viral proteins to the assembly site and the budding process. Subviral components such as the NP proteins of nucleocapsids, as well as the RNA of the influenza viral RNP and the matrix proteins, interact with the host cytoskeletal components during intracellular transport. Cytoskeletal components, particularly microtubules and microfilaments, are known to be involved in targeting proteins to apical and basolateral membrane domains. Actin filaments have been observed in budding paramyxoviruses, and are also present in released viral particles of both orthomyxo- and paramyxoviruses. These observations suggest that the host cytoskeletal elements are actively involved in the assembly and budding processes of virus particles. However, studies that attempted to examine these roles by using cytoskeletal disruptive agents like cytochalasins B and D have produced conflicting results. Some of these studies have reported drug-induced enhancement of virus assembly and release, while others found a decrease in or no effect on virus replication following treatment with these agents. It is likely that both the nature of host cells and viruses as well as the timing of the addition of these drugs in the infectious cycle may have contributed to the variable results obtained in these studies. For example, drugs added relatively early in the infectious cycle might interfere with transport of viral components and thereby inhibit virus assembly and release, whereas, if these drugs were added late in the infectious cycle, they might enhance virus release by disrupting microfilaments and facilitating closure of viral buds.
For paramyxoviruses, three viral components — namely, the NP of viral nucleocapsids (RNP), the M protein, and two transmembrane glycoproteins (F and HN) — are critically important for the assembly and morphogenesis of virus particles. However, the function of some other viral proteins such as P or L in the assembly process is unknown. These latter two proteins are synthesized independently from different viral messengers, and in different cellular compartments than NP, M, F and HN, and their synthesis appears to be temporally regulated. NP, the major component of the viral capsid, is critically required for both genome replication and nucleocapsid formation. Both free (soluble) and nucleocapsid-bound NP are present in the cytoplasm of virus-infected cells. Viral genome (vRNA) synthesis is coordinated with NP synthesis such that vRNA synthesis will not take place in the absence of a sufficient level of free NP proteins. Both the M and NP proteins are synthesized on free polyribosomes in the cytoplasm. NP associates with vRNA to form the RNP complex, and M proteins interact with the vRNP to form the M/vRNP complex. These proteins and complexes are formed in the cytoplasm. These M/NP (or M/RNP) complexes as well as M and NP proteins become associated independently with cytoskeletal components.
These components then interact with the F and HN glycoproteins either individually or together during their transport through the exocytic pathway or following insertion of F and HN into the plasma membrane, or both. The site of interaction between the M/RNP complex and the viral glycoproteins is essentially regulated by the presence and availability of these viral membrane glycoproteins in specific membrane components. For example, late in the infectious cycle in virus-infected cells, the majority of the viral glycoproteins are already present on the plasma membrane; therefore, the M/RNP–glycoprotein interaction is likely to occur predominantly on the plasma membrane. In addition, Sendai virus M protein can interact with Sendai virus glycoproteins in the absence of NP protein or RNP during exocytic transport of glycoproteins (
The assembly of orthomyxoviruses (influenza viruses,
After uncoating, the infecting vRNPs that have entered the cell are transported into the nucleus for replication and transcription of viral RNAs, following which the progeny vRNPs are formed in the nucleus. In order for assembly of progeny influenza viruses to occur, the newly formed vRNPs must be transported out of the nucleus into the cytoplasm and then directed to the assembly site on the plasma membrane. However, since the NP protein possesses nuclear localization and nuclear retention signals, it is not clear what enables the vRNP to exit the nucleus. Although it appears that dissociation of the incoming M1 matrix protein in the acid pH of endosomes during the viral uncoating process is necessary in order for the vRNPs to subsequently enter nucleus, massive association of M1 with progeny vRNP in the nucleus is not required for exit of vRNPs from the nucleus into the cytoplasm. A study performed in ts51 (temperature-sensitive M1 mutant WSN of influenza virus) virus-infected cells has revealed that progeny vRNPs exit from the nucleus into cytoplasm at the restrictive temperature when the ts51 M1 protein is not functional, and the majority of this mutant's newly synthesized M1 proteins is retained in the nucleus. The ts defect is due to hyperphosphorylation of the M1 protein at the restrictive temperature. Recently, NS2 protein has been shown to provide the nuclear export signal for exit of viral RNP from the nucleus into the cytoplasm (
The following scenario is envisioned for the nuclear exit of vRNP (
Once vRNPs are exported from the nucleus into the cytoplasm, the subsequent sequence of events leading to assembly and budding have yet to be resolved for orthomyxoviruses. Late in the infectious cycle, the majority of the cytoplasmic M1 proteins in virus-infected cells become membrane bound immediately after their synthesis. Pulse-chase experiments have shown that late in the infectious cycle the newly synthesized M1 protein incorporated quickly into the progeny virus particles, suggesting that M1 is a rate-limiting factor in virus morphogenesis and release. This result also suggests that viral RNPs and glycoproteins are already present on the plasma membrane of virus-infected cells late in the replication cycle at the time when M1, soon after its synthesis, interacts with the membrane-bound nucleocapsid complexes. However, the interaction of M1 with membrane-associated components is not totally dependent on having viral glycoproteins associated with the membrane. This independence has been demonstrated by the fact that free M1 alone can become membrane bound and, possibly, even diffuse to the assembly site, where it becomes stably associated with the membrane after it interacts with other previously bound molecules of M1, membrane glycoproteins, and vRNPs. However, despite this evidence, the precise role of the viral components (namely, M1, glycoproteins, and nucleocapsids), as well as the sequence of steps involved in the association of M1 with the membrane during influenza virus assembly and budding, have yet to be determined. Since progeny virus particles can be formed that lack either the HA or NA proteins or lack the cytoplasmic tail of the HA or NA, it would seem that the association of M1 with any one of these glycoproteins is sufficient to allow for viral morphogenesis and that M1 may interact with the transmembrane domain as well as cytoplasmic tail of either of these glycoproteins. On the other hand, since only a few molecules of M2 protein are found in the virus particle, M2 is unlikely to be a major factor in membrane association of M1 or in assembly and morphogenesis of virus particles.
Finally, how the eight different RNA (or RNP) segments are incorporated into each infectious virus particle remains unclear. Two models have been proposed: (a) selective assembly of eight unique vRNP segments, and (b) random assembly of multiple vRNP segments into a virus particle. The latter model would propose that more than eight RNA segments are incorporated randomly into each particle, so that a fraction of virus particles will possess eight separate RNA segments and be infectious. The majority of data from genetic and biochemical experiments does not differentiate between these two models. Selective assembly predicts that each virus particle will possess only eight separate RNA segments. However, extra vRNA segments have been shown to be present in virus particles under forced selection, suggesting that eight vRNA segments can be randomly incorporated into virus particles. This, however, may represent only a minority of virus particles. On the other hand, an extrapolation of findings that describe the loss of a homologous vRNA segment in defective interfering (DI) virus particles would suggest that multiple segments of the same RNA segment are not favored for incorporation into the same virus particle and would therefore favor some selective process for achieving incorporation of specific vRNP segments into a virion during viral assembly. However, there are as yet neither definitive data nor a specific hypothesis to explain the mechanism by which selective incorporation of vRNPs in a virus particle is accomplished. Finally, the pleomorphism and plasticity of virus particles (
Last, after budding from the host cell, viruses must be released into the surrounding medium and spread outward to infect other cells. Some viral components are critically involved in the viral release process. The data from ts viruses at restrictive temperature clearly demonstrate that the viral neuraminidase (NA) protein is involved in virus release. The NA removes the sialic acid, which is the receptor for influenza virus, from membrane glycolipids and glycoproteins of both the virus and the virus-infected cells, and thus prevents self-aggregation among virus particles and reattachment to the virus-infected cell.
The site and nature of budding can be an important contributory factor in viral pathogenesis, particularly for such respiratory viruses as the influenza and Sendai viruses. The influenza and Sendai viruses bud from the apical surface of polarized epithelial cells (e.g., bronchial epithelial cells) into the lumen of the lungs and are therefore usually pneumotropic, that is, restricted to the lungs, and do not cause viremia or invade other internal organs. However, occasionally, some influenza viruses — like fowl plague (H5 or H7) viruses (H5 or H7 indicates hemagglutinin subtype specificity of type A influenza viruses) and WSN (H1N1) viruses — are not restricted to the lungs and produce viremia infecting other internal organs (pantropism) and cause a high degree of mortality in infected animals. In humans, most of the influenza viruses are pneumotropic and do not spread to other internal organs. However, it is not clear if the Spanish Influenza of 1918, the most devastating influenza pandemic in recorded human history, which killed 20 to 40 million people worldwide, particularly affecting young healthy adults, was only pneumotropic. In addition to pneumonia, some people died due to massive pulmonary hemorrhage and edema (see
The severity of viral pathogenesis depends on both viral and host factors, including host immunity. The virulence determinants of influenza viruses are complex and multigenic. However, one factor that has been thought to be critical in viral growth and virulence is the cleavability of HA → HA1 and HA2. Influenza virus is normally restricted to the lungs because its HA can be cleaved by tryptase Clara, a serine protease restricted to the lungs. However, some HA that contains multiple basic amino acids at the HA1–HA2 junction, as is found only in H5 and H7 avian subtypes, can be cleaved by furin and subtilisin-type enzymes, which are present ubiquitously. Therefore, such viruses can grow in other organs. In addition, the NA of some influenza viruses (e.g., WSN viruses) binds and activates plasminogen into plasmin in the vicinity of HA, and the activated plasmin cleaves HA→ HA1 and HA2, rendering the virus infectious. Therefore, WSN virus, which lacks multiple basic residues in its HA, can grow and multiply in tissues other than the lungs.
However, although the cleavage of HA→ HA1 and HA2 is a major virulence factor, it is not the only factor contributing to the pantropism of a normally pneumotropic flu virus. For example, although WSN virus is pantropic and neurovirulent in the mouse, gene reassortment experiments demonstrated that the WSN NA gene responsible for the cleavage of HA was not sufficient for neurovirulence in chickens or mice. Other WSN genes, like the M and NS genes, in addition to the NA gene, were required for neurovirulence and, therefore, likely pantropism. The function of M and NS genes in neurovirulence is not known. The M gene in Sendai virus has been shown to affect apical versus basolateral budding and contribute to the pantropism of F1-R Sendai virus mutant (
Sendai virus, like influenza virus, is a pneumotropic mouse virus that buds apically. However, a Sendai virus mutant, F1-R, which exhibited pantropism possessed two characteristics (
The replication and morphogenesis processes of viruses are different from those of either prokaryotic or eukaryotic organisms. In this chapter, I have presented some of the general steps involved in the viral infectious cycle, including: entry, uncoating, transcription, translation, replication, and assembly processes, and the possible role of budding in viral pathogenesis. Of these, viral morphogenesis is the most obscure phase in the virus life cycle. Yet, knowledge of how the particles are formed during this morphogenetic stage is fundamental to understanding virus growth and multiplication, and therefore is crucial in defining viral infectivity, transmission, virulence, tissue tropism, host specificity, and pathogenesis, and contributes to an overall understanding of the disease process and progression of disease, including host morbidity and mortality. In addition, the site of budding can affect virus virulence and pathogenesis. Elucidation of the viral replicative and assembly processes is critical in terms of enabling us to find ways to block these steps and thereby intervene in the viral life cycle and disease process. Much remains to be done to achieve these goals, particularly in terms of elucidating those stages of the viral assembly process that relate to how viral components are brought to the assembly site, how those components interact with each other at the assembly site, and how viral budding actually occurs. A better understanding of viral replication and morphogenesis may lead us to develop novel therapeutic agents capable of interfering with these critical steps in viral multiplication and pathogenesis.
Research in the author's laboratory was supported by grants from the National Institutes of Health. The author thanks Kiet Chi Tran for drawing some of these figures. The source for the other figures is gratefully acknowledged in the captions. The author is grateful to Dr. Felix Wettstein for helpful suggestions and Eleanor Berlin for typing the manuscript.