51 Replication of viruses
Unlike bacteria, which can grow on inert media, viruses are obligate intracellular parasites and can multiply only in viable cells. This requirement arises from their limited genomic composition which obliges them to utilize host cell organelles, enzymes and other macromolecules for replication. The effects of viral multiplication on host cells range from minor changes in cellular metabolism to cytolysis.
The replicative cycle of a virus can be conveniently divided into a number of stages (Box 51.1). A virion must first attach to cell surface receptors in order to produce infection. Initial virus–cell interaction is a random event which relates to the number of virus particles present and the availability of appropriate receptor molecules. Virus–cell interaction determines both the host range and the tissue tropism of viral species. Viruses have evolved to the point where they can utilize a wide range of host cell surface proteins as receptors. Many of these surface molecules are highly conserved and are essential for fundamental cellular functions. Some viruses have more than one type of ligand molecule and they may bind to several cell surface receptors
in sequential order during attachment. Some viral species can detach and adsorb to another cell when infection of a particular host cell does not proceed. Detachment of orthomyxoviruses and paramyxoviruses from host cells is mediated by viral neuraminidase, a receptor-destroying enzyme.
Virus uptake or penetration of the cell's plasma membrane is an energy-dependent process that can occur in two main ways, endocytosis or direct entry. Several endocytic mechanisms are described including clathrin-mediated, caveolar/raft-mediated and macropinocytosis. Acidification within the endosome leads to structural changes in the internalized virus that facilitate its entry into the cytosol. The envelopes of some viruses, such as orthomyxoviruses, rhabdoviruses and flaviviruses, fuse with the membrane of endosomes, releasing nucleocapsids directly into the cytoplasm. An alternative strategy which is used by some enveloped viruses, including paramyxoviruses, retroviruses and herpesviruses, involves fusion of the viral envelope with the plasma membrane. This allows release of the nucleocapsid directly into the host cell cytoplasm. The entry of some non-enveloped viruses such as picornaviruses involves the direct passage or translocation of viral genomes into the cytoplasm through channels or pores in the plasma membrane.
Uncoating is the process whereby the viral genome is released in a form suitable for transcription. In the case of enveloped viruses, in which the nucleocapsid is discharged directly into the cytoplasm, transcription can usually proceed without complete uncoating. For other viruses, uncoating results from disruption of the capsid protein–nucleic acid relationship as a result of proteolytic enzyme activity or following binding to particular ‘replication’ sites. In reoviruses, the genome may express all functions without complete release from the capsid. For the majority of non-enveloped viruses complete uncoating occurs. Poxviruses are uncoated in two stages. The initial stage is mediated by host enzymes, with complete release of viral DNA from the core requiring virus-specified proteins. In some viruses which replicate in the cell nucleus, uncoating may be completed at the nuclear pore complex.
The synthesis of viral proteins by host cells, which is the central event in replication of viruses, requires the production of viral mRNA. While DNA viruses which replicate in the nucleus can employ host cell transcriptases to synthesize viral mRNA, other viruses use their own enzymes to generate mRNA. Viruses have evolved strategies which facilitate interference with the activity of cellular mRNA. Viruses direct the synthesis of either a separate mRNA for each gene or mRNA encompassing several genes. Eukaryotic cell protein-synthesizing mechanisms, however, translate only monocistronic messages. If a large precursor protein molecule is produced, cleavage into individual proteins is required and each family of viruses employs unique strategies for this purpose.
Based on the nature of the genome and the method of mRNA synthesis, viruses of veterinary importance can be grouped into seven classes, the Baltimore classification. Central to this scheme is the designation of the genome of single-stranded RNA viruses as positive-sense or negative-sense nucleic acid. In this context, the word ‘sense’ refers to nucleic acid polarity. The nucleic acid of positive-sense single-stranded RNA viruses is mRNA in sense and can be translated directly on host ribosomes, forming viral proteins.
Replication of DNA viruses
Double-stranded DNA viruses, such as herpesviruses, papovaviruses and adenoviruses that replicate in the nucleus of the cell, have a relatively direct replication strategy. The viral DNA is transcribed by cellular DNA-dependent RNA polymerase (transcriptase), forming mRNA. In contrast, the single-stranded DNA viruses, parvoviruses and circoviruses, which also replicate in cell nuclei, utilize cellular DNA polymerase to synthesize double-stranded DNA. This is then transcribed to mRNA by cellular transcriptases. Parvoviruses and circoviruses require dividing cells for their replication. The replication of large DNA viruses (poxviruses and African swine fever virus), which encode all enzymes required for replication, occurs primarily in the cytoplasm.
A defined temporal sequence of events occurs during transcription and replication of DNA viruses. Specified genes encode for early proteins, which include the enzymes and other proteins necessary for virus replication and for suppression of the synthesis of host cell proteins. Subsequently, replication of viral nucleic acid and transcription of the genes which encode the late proteins occur. These late proteins, which are also often transcribed from newly formed viral nucleic acid, are structural components synthesized late in the infection cycle. This temporal sequence is not clearly demonstrable in the replicative cycles of RNA viruses in which most of the genetic information is expressed contemporaneously.
Replication of RNA viruses
Reoviruses and birnaviruses, double-stranded RNA viruses, have segmented genomes. Transcription occurs in the cytoplasm under the direction of a viral transcriptase. The negative-sense strand of each segment is transcribed to produce individual mRNA molecules. In contrast, the genomes of positive-sense single-stranded RNA viruses can act directly as mRNA after infection. The enzymes necessary for genome replication in these viruses are produced after infection by direct translation of virion RNA. This RNA can bind directly to ribosomes and is translated to yield a single polyprotein which is then cleaved to yield both functional and structural proteins. Because direct translation can occur, naked RNA extracted from such viruses is infectious. The positive-sense single-stranded RNA viruses utilize a number of different synthetic pathways during replication. In togaviruses, only about two-thirds of the viral RNA is directly translated during the first round of protein synthesis. Subsequently, full-length negative-sense RNA is synthesized and, from this, a full-length positive-sense RNA destined for encapsidation and a one-third length positive-sense RNA strand are formed. The genomes of caliciviruses, coronaviruses and arteriviruses also encode for mRNA which can be partial or full length.
Negative-sense single-stranded RNA viruses possess an RNA-dependent RNA polymerase. The naked RNA of these viruses, unlike that of the positive-sense single-stranded RNA viruses, cannot initiate infection. After infection by the virion, the genomic RNA functions as a template for transcription of positive-sense mRNA and also for virus replication, utilizing the same polymerase. The positive-sense RNA subsequently serves as the template for synthesis of negative-sense genomic RNA. Most single-stranded negative-sense RNA viruses replicate in the cytoplasm of the cell. Notable exceptions are orthomyxoviruses and Borna disease virus, which replicate in the nucleus. Part of the segmented genome of some members of the Bunyaviridae is ambisense, utilizing a mixed replication strategy with features characteristic of both positive-sense and negative-sense single-stranded RNA viruses.
The genome of retroviruses consists of positive-sense single-stranded RNA which does not function as mRNA. Instead, a single-stranded DNA copy is produced by RNA-dependent DNA polymerase (reverse transcriptase) using the viral RNA as a template. As the second strand of DNA is formed, the parental RNA is removed from the RNA–DNA hybrid molecule. The double-stranded DNA is integrated into the host cell genome as a provirus and can subsequently be transcribed to new viral RNA.
Protein synthesis
Within the cell, the sites at which particular proteins are synthesized relate to the type and function of the protein. Membrane proteins and glycoproteins are synthesized on membrane-bound ribosomes, while soluble proteins including enzymes are synthesized on ribosomes free in the cytoplasm. Short specific amino acid sequences, known as sorting sequences, facilitate the incorporation of proteins at various cellular locations where they are required for metabolic activity. Most viral proteins undergo post-translational modification including proteolytic cleavage, phosphorylation and glycosylation. During glycosylation, sugar side-chains are added to viral proteins in a programmed manner as the proteins are being transferred from the rough endoplasmic reticulum to the Golgi apparatus. This event occurs in preparation for the final assembly of intact virions prior to their release from the cell.
Assembly and release of virions
The mechanisms required for the assembly and release of enveloped and non-enveloped viruses are distinct. Non-enveloped viruses of animals have an icosahedral structure. The structural proteins of these viruses associate spontaneously in a symmetrical and stepwise fashion, forming procapsids. Subsequently, viral nucleic acid is incorporated into the procapsid. Proteolytic cleavage of specific procapsid polypeptides may be required for the final formation of infectious particles. Non-enveloped viruses are usually released following cellular disintegration. The assembly of picornaviruses and reoviruses occurs in the cytoplasm of the cell, whereas parvoviruses, adenoviruses and papovaviruses are assembled in the nucleus.
The final step in the process of virion assembly for enveloped viruses involves acquisition of an envelope by budding from membranes of the cell. Prior to budding, cell membranes are modified by the insertion of virus-specified transmembrane glycoproteins which aggregate in patches in the plasma membrane. Their presence alters the antigenic composition of infected cells which become targets for cytotoxic T lymphocytes. In the case of icosahedral viruses, the proteins of their nucleocapsids bind to the hydrophilic domains of the virus-specified membrane glycoprotein spikes which project slightly into the cytoplasm. As a result, the nucleocapsid becomes surrounded by the altered portion of membrane during budding. The nucleocapsids of helical viruses bind to a virus-specified matrix (M) protein which in turn binds to the hydrophilic domains of the virus-specified glycoproteins that line the cytoplasmic side of membrane patches.
Budding of viruses through the plasma membrane usually does not breach the integrity of the membrane and, as a result, many enveloped viruses are non-cytopathic and may be associated with persistent infections. However, unlike most other enveloped viruses, togaviruses, paramyxoviruses and rhabdoviruses are cytolytic. Flaviviruses, coronaviruses, arteriviruses and bunyaviruses acquire their envelopes inside cells by budding through the membranes of the rough endoplasmic reticulum or the Golgi apparatus. These viruses are then transported in vesicles to the cell surface where the vesicle fuses with the plasma membrane releasing the virion by exocytosis. Herpesviruses, which replicate in the nucleus, are unique in that they bud through the inner lamella of the nuclear membrane and accumulate in the space between inner and outer lamellae, in the cisternae of the endoplasmic reticulum and in cytoplasmic vesicles.
Release from the cell occurs either by exocytosis or by cytolysis. Epithelial cells exhibit polarity and infecting viruses show a tendency to bud from either the apical surface which facilitates shedding from the host or from the basolateral surface which facilitates systemic spread. The assembly and release of poxviruses is a complex process taking several hours. Although replication occurs entirely in the cytoplasm of the host cell at discrete sites, termed viroplasms or ‘viral factories’, nuclear factors may be involved in transcription and assembly. Maturation leading to the formation of infectious intracellular mature virus follows. Virus particles then move out of the assembly area and become enveloped in a double membrane derived from the trans-Golgi network. At the periphery of the cell, fusion with the plasma membrane results in loss of the outer layer of the double membrane and release of extracellular enveloped virus.