Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 278

Viral RdRPs must select viral templates from among a vast excess of cellular mRNAs and then initiate correctly to ensure accurate RNA synthesis. Different mechanisms that contribute to template specificity have been identified. Initiation specificity may be regulated by the affinity of the RdRP for the initiating nucleotide. For example, the RdRPs of bovine viral diarrhea virus and bacteriophage ϕ6 prefer 3′-terminal C. Reovirus RdRP prefers a G at the second position of the template RNA. This preference is controlled by hydrogen bonding of carbonyl and amino groups of the G with two amino acids of the enzyme. Both preferences would exclude initiation on cellular mRNAs, the great majority of which end in poly(A).

Template specificity may also be conferred by the recognition of RNA sequences or structures at the 5′ and 3′ ends of viral RNAs by viral proteins. RNA synthesis initiates specifically within a polypyrimidine tract in the 3′ untranslated region of hepatitis C virus RNA. The 3′ noncoding region of polioviral genomic RNA contains an RNA pseudoknot structure that is conserved among picornaviruses (Fig. 6.9). A viral protein (3AB-3CD) binds this structure and may direct the RdRP to that site for the initiation of (−) strand RNA synthesis. The precursor to poliovirus 3CD^pro plays an important role in viral RNA synthesis by participating in the formation of a ribonucleoprotein at the 5′ end of the (+) strand RNA. This protein, together with cellular poly(rC)-binding protein 2, binds to a cloverleaf structure in the viral RNA (Fig. 6.9). Alterations within the RNA-binding domain of 3CD inhibit binding to the cloverleaf and RNA synthesis.

Internal RNA sequences may also confer initiation specificity to RdRPs. The cis-acting replication elements (cre) in the coding sequence of poliovirus protein 2C and rhinovirus capsid protein VP1 contain short RNA sequences that are required for RNA synthesis. These sequences are binding sites for 3CD^pro and, as discussed previously, serve as a template for uridylylation of the VPg protein (Fig. 6.9).

During mRNA synthesis by influenza virus polymerase, sequences at the RNA termini ensure that the 5′ ends of newly synthesized viral mRNAs are not cleaved and used as primers (Fig. 6.12). If such cleavage were to occur, there would be no net synthesis of viral mRNAs. Polymerase binding to two sites in the genomic RNA blocks access of a second P protein and protects newly synthesized viral mRNA from endonucleolytic cleavage by P proteins.

Protein-protein interactions can also direct RdRPs to the RNA template. The vesicular stomatitis virus RdRP for mRNA synthesis consists of the P protein and the L protein, the catalytic subunit. The P protein binds both the L protein and the ribonucleoprotein containing N and the (−) strand RNA. In this way, the P protein brings the L protein to the RNA template [see “(−) Strand RNA” below]. Cellular general initiation proteins have a similar function in bringing RNA polymerase II to the correct site to initiate transcription of DNA templates.

While viral RdRPs copy only viral RNAs in the infected cell, purified polymerases often lack template specificity. The replication complex in the infected cell may contribute to template specificity by concentrating reaction components to create an environment that copies viral RNAs selectively. Replication of viral RNAs on membranous structures might contribute to such specificity (Chapter 14).