Translation

Capped, polyadenylated, and processed monocistronic viral mRNAs bind to ribosomes and are translated into protein in the same fashion as cellular mRNAs. The sequence of events has been closely studied for reovirus Each monocistronic mRNA molecule binds via its capped 5' terminus to the 40 S ribosomal subunit, which then moves along the mRNA molecule until stopped at the initiation codon The 60 S ribosomal subunit then binds, together with methionyl-transfer RNA various initiation factors, after which translation proceeds.

In mammalian cells, mRNA molecules are monocistronic (encoding only one protein), and, with few exceptions, translation commences only at the 5' initiation codon. However, with certain viruses polycistronic mRNA can be translated directly into its several gene products as a result of initiation, or reinitiation, of translation at interna! AUG start codons.

Where initiation of translation at an internal AUG is an option, a frame-shift can occur. Another mechanism, known as ribosomal frameshilting, occurs fortuitously when a ribosome happens to slip one nucleotide forward or back along an RNA molecule. This phenomenon is exploited by retroviruses to access the reverse transcriptase reading frame within the gag-jwl mRNA. Thus, considering also the phenomena of RNA splicing and RNA editing described earlier, it can be seen that there are several mechanisms of exploiting overlapping reading frames to maximize the usage of the limited coding potential of the small genomes of viruses.

Most viral proteins undergo posttranslational modifications such as phosphorylation (for nucleic acid binding), fatty acid acylation (for membrane insertion), glycosylation, or proteolytic cleavage (see below). Newly synthesized viral proteins must also be transported to the various sites in the cell where they are needed, for example, back into the nucleus in the case of viruses that replicate there. The sorting signals that direct this traffic are only beginning to be understood, as arc the polypeptide chain binding proteins ("molecular chaperones") that regulate folding, translocation, and assembly of oligomers of viral as well as cellular proteins.

Glycosylation of Envelope Proteins

Viruses exploit cellular pathways normally used for the synthesisof membrane-inserted and exported secretory glycoproteins (Fig. 3-7) The programmed addition of sugars occurs sequentially as the protein moves in vesicles progressively from the rough endoplasmic reticulum to the Golgi complex and then to the plasma membrane. The side chains of viral envelope glycoproteins are generally a mixture of simple ("high manno.se") and complex oligosaccharides, which are usually N-linked to asparagine but less commonly O-linked to serine or threonine. The precise composition of the oligosac-

Plasma membiane

Ribosome

Cytoplasm

Fig. 3-7 Glycvsylabor) of viral protein The amino terminus of viral envelope proteins initially contains a sequence of 15-30 hydrophobic amino acids, known as a signal sequence, which facilitates binding ot the growing polypeptide chain (dotted) to a receptor site on the cytoplasmic side of the rough endoplasmic reticulum and its passage through the lipid bilayer to the luminal side Oligosaccharides are then added in N-linkage to certain asparagine residues of the nascent polypeptide by en Woe transfer of a mannose-rich "core" of preformed oligosaccharides, and glucose residues are removed by glycosidases ("trimming") The viral glycoprotein is then transported from tfie rough endoplasmic reticulum to the Golgi complex Heic the core carbohydrate is further modified by the removal of several mannose residues and the addition of further N acelylglucosamine, galactose, and the terminal sugar, sialic acid or fucose The completed side chains are a mixture of simple ("high mannose") and complex oligosaccharides A coated vesicle then transports the completed glycoprotein to the cellular membrane from which the particular virus buds.

Golgi apparatus

Plasma membiane h

Ribosome

Cytoplasm

Fig. 3-7 Glycvsylabor) of viral protein The amino terminus of viral envelope proteins initially contains a sequence of 15-30 hydrophobic amino acids, known as a signal sequence, which facilitates binding ot the growing polypeptide chain (dotted) to a receptor site on the cytoplasmic side of the rough endoplasmic reticulum and its passage through the lipid bilayer to the luminal side Oligosaccharides are then added in N-linkage to certain asparagine residues of the nascent polypeptide by en Woe transfer of a mannose-rich "core" of preformed oligosaccharides, and glucose residues are removed by glycosidases ("trimming") The viral glycoprotein is then transported from tfie rough endoplasmic reticulum to the Golgi complex Heic the core carbohydrate is further modified by the removal of several mannose residues and the addition of further N acelylglucosamine, galactose, and the terminal sugar, sialic acid or fucose The completed side chains are a mixture of simple ("high mannose") and complex oligosaccharides A coated vesicle then transports the completed glycoprotein to the cellular membrane from which the particular virus buds.

charides (glycans) is determined not only by the amino acid sequence and tertiary structure of the proteins concerned, but more importantly by the particular cellulaT glycosyltransferases prevalent in the type of cell in which the virus happens to be growing at the time.

Posttranslational Cleavage of Proteins

In the case of the plus sense picornaviruses and flaviviruses, the polycistronic viral RNA is translated directly into a single polyprotein which carries proteinase (protease) activity that cleaves the polyprotein at defined recognition sites into smaller proteins. The first cleavage steps are carried out while the polyprotein is still associated with Ihe ribosome. Some of the larger intermediates exisl only fleetingly; others are functional tor a short period but are subsequently cleaved by additional virus-coded proteases to smaller proteins wilh alternative functions. Posttranslational cleavage occurs in several other RNA virus families, for example, togaviruses and caliciviruses, in which poly-proteins corresponding to large parts of the genome are cleaved. Some viruses encode several different proteases. Most are either trypsin-Iike (serine or cysteine proteases), pepsin-like (aspartyl proteases), or papain-like (thiol proteases).

Cellular proteases, present in particular organelles such as the Golgi complex or transport vesicles, are also vital to the maturation and assembly of many viruses. For example, cleavage of the hemagglutinin glycoprotein of orthomyxoviruses or the fusion glycoprotein of paramyxoviruses is essential for the production of infectious virions

Classes of Viral Proteins

Table 3-3 lists the various classes of proteins encoded by the genomes of viruses. In general, the proteins translated from the early transcripts of DNA viruses include enzymes and other proteins required for the replication of viral nucleic acid, as well as proteins that suppress host cell RNA and protein synthesis. The large DNA viruses (poxviruses and herpesviruses) also encode a number of enzymes involved in nucleotide metabolism.

The late viral proteins are translated from late niRNAs, most of which are transcribed from progeny viral nucleic acid molecules Most of the late proteins are viral structural proteins, and they are often made in considerable excess

Some viral proteins, including some with other important functions, serve as regulatory proteins, modulating the transcription or translation of cellular genes or of early viral genes The large DNA viruses also encode numerous additional proteins, sometimes called vimkmes, which do not regulate the viral replication cycle itself but influence the host response to infection (see Chapter 7).

Table 3-3

Categories of Proteins Encoded by Viral Genomes

Structural proteins of (he vinon' Virion-assocrated enzymes, especially transcriptase1.

Nonstructural proteins, mainly enzymes, required for transcription, replication of viral nucleic acid, and cleavage of proteins' Regulatory proteins which control the temporal sequence of expression of the viral genome'' Proteins down-regulating expression of cellular genes1

Oncogene products (oncoproteins) and inactivators of cellular tumor suppressor proteins' Proteins influencing viral virulence, host range, tissue tropism, etc v

Virokines.«, which acl on noninfected cells to modulate the progress of infection in the body as a whole''

" Comprising rapsid and (for some viruses) core and/or envelope RNA viruses of plus sense and nuclear DNA viruses do not carry a transcriptase in the virion Virions of some viruses, e g , poxviruses, also contain several other enzymes ' DNA and RNA polymerases, helicases, proteases, etc DNA viruses with large complex genomes, notably poxviruses and herpesvnuses, n,lso encode numerous enzymes needed for nucleotide synthesis

'' Site-specific DNA-binding proteins (transcription farlors) which hind to enhancer sequences in the viral genome, or to another transcription factor Some may act in fruits (transactivatnrs) ' Usually by inhibiting transcription, sometimes translation

' Upgrade expression of certain cellular genes; may lead to cell transformation and eventually to cancer, as observed with herpesviruses, adenoviruses, papovavnuses, and retroviruses < Virokiries have been recorded so far mainly in the more complex DNA viruses (poxviruses, herpesviruses, adenoviruses) but may be more widespread. '' Virokmes act mainly by subverting the immune response by inhibiting cytokines, down-regulaling MI1C expression, blocking the complement cascade, etc (see Table 7 2)

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