Principles of Virology. Jane Flint

are translated upon entry into the cytoplasm to produce viral proteins, including the RdRP and accessory proteins. The (+) strand RNA genome is copied to a (−) strand, which in turn is used as a template for the synthesis of additional (+) strands (Fig. 6.1). Newly synthesized (+) strand RNA molecules can serve as templates for further genomic replication, as mRNAs for the synthesis of viral proteins, or as genomic RNAs to be packaged into progeny virions. Because picornaviral mRNA is identical in sequence to the viral RNA genome, all RNAs needed for the reproduction of these viruses can be made by a simple set of RNA synthesis reactions (Fig. 6.1). Such simplicity comes at a price, however, because synthesis of individual viral proteins cannot be regulated. However, polioviral gene expression can be controlled by the rate and extent of polyprotein processing. For example, the precursor of the viral RdRP, 3CD, cannot polymerize RNA, but is a protease that cleaves at certain Gln-Gly amino acid pairs in the polyprotein. Therefore, regulating the processing of the precursor 3CD controls the concentration of RNA polymerase.

The mechanisms of mRNA synthesis of other (+) strand RNA viruses allow structural and nonstructural proteins (generally needed in greater and lesser quantities, respectively) to be made separately. The nonstructural proteins are synthesized from full-length (+) strand (genomic) RNA, while structural proteins are translated from subgenomic mRNA(s). This strategy is characteristic of the replication cycles of coronaviruses, caliciviruses, and alphaviruses. Translation of the Sindbis virus (+) strand RNA genome yields the nonstructural proteins that synthesize a full-length (−) strand (Fig. 6.16). The (–) strand RNA molecules contain not only a 3′-terminal sequence for initiation of (+) strand RNA synthesis, but also an internal initiation site, used for production of a 26S subgenomic mRNA.

Alphaviral genome and mRNA synthesis is regulated by the sequential production of three RNA polymerases with different template preferences. All three enzymes are derived from the nonstructural polyprotein P1234 and contain the complete amino acid sequence of this precursor (Fig. 6.17). The covalent connections among the segments of the polyprotein are successively broken, with ensuing alterations in the specificity of the enzyme. It seems likely that each proteolytic cleavage induces a conformational change in the RdRP that alters its template specificity.

      The mRNAs synthesized during infection by most RNA viruses contain a 3′ poly(A) sequence, as do the vast majority of cellular mRNAs (exceptions are mRNAs of arenaviruses and reoviruses). The poly(A) sequence is encoded in the genome of (+) strand viruses. For example, polioviral (+) strand RNAs contain a 3′ stretch of poly(A), approximately 62 nucleotides in length, which is required for infectivity. The (−) strand RNA contains a 5′ stretch of poly(U), which is copied to form this poly(A).

Figure 6.16 Genome structure and expression of an alphavirus, Sindbis virus. The 11,703-nucleotide Sindbis virus genome contains a 5′-terminal cap structure and a 3′ poly(A) tail. A conserved RNA secondary structure at the 3′ end of (+) strand genomic RNA is thought to control the initiation of (−) strand RNA synthesis. At early times after infection, the 5′ region of the genomic RNA (nonstructural open reading frame [ORF]) is translated to produce two nonstructural polyproteins: P123, the synthesis of which is terminated at the first translational stop codon (indicated by the box); and P1234, produced by an occasional (15%) read-through of this stop codon. The P1234 polyprotein is proteolytically cleaved to produce the enzymes that catalyze the various steps in genomic RNA replication: the synthesis of a full-length (−) strand RNA, which serves as the template for (+) strand synthesis, and either full-length genomic RNA or subgenomic 26S mRNA. The 26S mRNA, shown in expanded form, is translated into a structural polyprotein (p130) that undergoes proteolytic cleavage to produce the virion structural proteins. The 26S RNA is not copied into a (−) strand because a functional initiation site is not formed at the 3′ end.

Synthesis of Nested Subgenomic mRNAs

An unusual pattern of mRNA synthesis occurs in cells infected with members of the families Coronaviridae and Arteriviridae, in which subgenomic mRNAs that form a 3′-coterminal nested set with the viral genome are synthesized (Fig. 6.18). These viral families were combined into the order Nidovirales to denote this shared property (nidus is Latin for “nest”).

Figure 6.17 Three RNA polymerases with distinct specificities in alphavirus-infected cells. These RdRPs contain the entire sequence of the P1234 polyprotein and differ only in the number of proteolytic cleavages in this sequence.

Figure 6.18 Nidoviral genome organization and expression. (A) Organization of open reading frames. The (+) strand viral RNA is shown at the top, with open reading frames as boxes. The genomic RNA is translated to form polyproteins 1a and 1ab, which are processed to form the RdRP. Structural proteins are encoded by nested mRNAs. (B) Model of the synthesis of nested mRNAs. Discontinuous transcription occurs during (−) strand RNA synthesis. Most of the (+) strand template is not copied, probably because it loops out as the polymerase completes synthesis of the leader RNA (orange). The resulting (−) strand RNAs, with leader sequences at the 3′ ends, are then copied to form mRNAs.

      The subgenomic mRNAs of these viruses comprise a leader and a body that are synthesized from noncontiguous sequences at the 5′ and 3′ ends, respectively, of the viral (+) strand genome (Fig. 6.18A). The leader and body are separated by a conserved junction sequence encoded both at the 3′ end of the leader and at the 5′ end of the mRNA body. Subgenome-length (−) strands are produced when the template loops out as the polymerase completes synthesis of the leader RNA (Fig. 6.18B). These (−) strand subgenome-length RNAs then serve as templates for mRNA synthesis.

(−) Strand RNA

The genes of RNA viruses with a nonsegmented (−) strand RNA genome are expressed by the production of subgenomic mRNAs in infected cells (Fig. 6.19). An RdRP composed of one molecule of L protein associated with four molecules of P protein is thought to carry out vesicular stomatitis virus mRNA synthesis. Individual mRNAs are produced by a series of initiation and termination reactions as the RdRP moves down the viral genome (Fig. 6.20). This start-stop mechanism accounts for the observation that 3′-proximal genes must be copied before downstream genes (Box 6.3). The viral RdRP is unable to initiate synthesis of each mRNA independently.

Figure 6.19 Vesicular stomatitis viral RNA synthesis. Viral (−) strand genomes are templates for the production of either subgenomic mRNAs or full-length (+) strand RNAs. The switch from mRNA synthesis