Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 282

The genome and mRNA of some (+) strand RNA viruses are identical. The genome RNAs of the Picornaviridae and Flaviviridae 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.