A general picture of RNAdirected RNA replication

With the exception of retroviruses and some unusual viruses related to viroids, single-stranded (ss) RNA virus genome replication requires two stages; these are shown in Fig. 14.1(b). First, the input strand must be transcribed (using Watson—Crick base-pairing rules) into a strand of complementary sequence and opposite polarity. Replication occurs as a "fuzzy," multi-branched structure. This complex, dynamic structure contains molecules of viral transcriptase (replicase), a number of partially synthesized product RNA strands ("nascent" strands), and the genome-sense template strand. The whole ribonucleoprotein (RNP) complex is termed the type 1 replicative intermediate or RI-1. The single-stranded products generated from RI-1 are antisense to the genomic RNA.

This complementary strand RNA serves as a template for the formation of more genomic-sense RNA strands. This second replicative intermediate (RI-2) is essentially the same in

Fig. 14.1 Some general features of viruses containing RNA genomes that use RNA-directed RNA transcription in their replication. (a) The general relationship between viruses containing a genome that can be translated as the first step in the expression of viral genes versus those viruses that first must carry out transcription of their genome into mRNA utilizing a virion-associated transcriptase. (b) The basic rules for RNA-directed RNA replication. As with DNA-directed RNA and DNA synthesis, the new (nascent) strand is synthesized 5' to 3' antiparallel to the template, and the Watson—Crick base-pairing rules are the same, with U substituting for T. However, the very high thermal stability of dsRNA leads to complications. The major complication is that newly synthesized RNA must be denatured and removed from the template strand to avoid its "collapsing" into a double-stranded form. Formation of such dsRNA is an effective inducer of interferon (see Chapter 8, Part II), and it appears to be refractory to serving as a template when free in the cytoplasm. A second complication is that in order to generate a ssRNA molecule of the same coding sense as the virion genome, two replicative intermediates (RIs) must be generated. These intermediates are dynamic structures of ribonucleoprotein containing a full-length template strand, and a number of newly synthesized product RNA molecules growing from virion-encoded replicase that is traversing the template strand. RI-1 generates RNA complementary to the virion genomic RNA. This serves as a template for new virion genome RNA in RI-2.

Translation +

Viral protein

Virion transcriptase mRNA Translation Viral protein dsRNA

Virion transcriptase mRNA Translation Viral protein

Virion genomic RNA Replicase

RI-1

Replicase ^r^

Opposite sense to genomic RNA

Replicase ^r^

RI-2

Genomic sense RNA

New genomic sense RNA

structure as RI-1, except that the template strand is of opposite sense to genomic RNA and the nascent product RNA molecules are of genome sense. Remember:

  • Virion RNA is the template in RI-1.
  • RI-1 produces template RNA of opposite sense to virion RNA.
  • RNA that is complementary to virion RNA is the template in RI-2.
  • RI-2 is the intermediate for expression of RNA of the same sense as the virion.

One further general feature of the replication of RNA viruses is worth noting. The error frequency (i.e., the frequency of incorporating an incorrect base) of RNA-directed RNA replication is quite high compared to that for dsDNA replication. Thus, typically DNA-directed DNA replication leads to incorporation of one mismatched base per 107 to 109 base pairs, while RNA-directed RNA synthesis typically results in one error per 105 bases. Indeed, the error rate in the replication of some RNA genomes can be as high as one error per 104 nucleotides.

Part of the reason for this error rate for RNA is that there is no truly double-stranded intermediate; therefore, there is no template for error correction or "proofreading" of the newly synthesized strand as there is in DNA replication. A second reason is that RNA polymerases using RNA templates seem to have an inherently higher error frequency than those utilizing DNA as a template.

For these reasons, infection of cells with many RNA viruses is characterized by the generation of a large number of progeny virions bearing a few or a large number of genetic differences from their parents. This high rate of mutation can have a significant role in viral pathogenesis and evolution; further, it provides the mechanistic basis for the generation of defective virus particles described in Chapter 21. Indeed, many RNA viruses are so genetically plastic that the term quasi-species swarm is applied to virus stocks generated from a single infectious event, as any particular isolate will be, potentially at least, genetically significantly different from the parental virus. The concept of a quasi-species, as applied to virus populations, has been important for the application of evolutionary models to such populations. As a result, the analysis of mutational changes over time can employ the models that are used in population genetics.

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