ATP is required for initiation of poliovirus RNA synthesis in vitro: demonstration of tyrosine-phosphate linkage between in vitro-synthesized RNA and genome-linked protein

104

A molecular genetic analysis has been combined with an in vitro biochemical approach to define the functional interactions required for nucleotidyl protein formation during poliovirus RNA synthesis. A sitedirected lesion into the hydrophobic domain of a viral membrane protein produced a mutant virus that is defective in RNA synthesis at 39°C. The phenotypic expression of this lesion affects initiation of RNA synthesis, in vitro uridylylation of the genome-linked protein (VPg), and the in vivo synthesis of plus-strand viral RNAs. Our results support a model that employs a viral membrane protein as carrier for VPg in the initiation of plusstrand RNA synthesis. Our data also suggest that a separate mechanism could be used in the initiation of minusstrand RNA synthesis, thereby providing a means for strand-specific regulation of picornavirus RNA replication.

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Role of a viral membrane polypeptide in strand-specific initiation of poliovirus RNA synthesis

Giachetti

Semler

1991

104

“…The involvement of these proteins in RNA replication has been confirmed by genetic studies analyzing several mutants of 2B (17) and 2C (22,27,32,45). In addition, VPg (3B) or its precursor (3AB) has been proposed to function as a primer for the initiation of RNA polymerization (29,44).…”

mentioning

confidence: 95%

RNA duplex unwinding activity of poliovirus RNA-dependent RNA polymerase 3Dpol

Cho

Richards

Dmitrieva

et al. 1993

The ability of highly purified preparations of poliovirus RNA-dependent RNA polymerase, 3DPo1, to unwind RNA duplex structures was examined during a chain elongation reaction in vitro. Using an antisense RNA prehybridized to an RNA template, we show that poliovirus polymerase can elongate through a highly stable RNA duplex of over 1,000 bp. Radiolabeled antisense RNA was displaced from the template during the reaction, and product RNAs which were equal in length to the template strand were synthesized. Unwinding did not occur in the absence of chain elongation and did not require hydrolysis of the 'y-phosphate of ATP. The rate of elongation through the duplex region was comparable to the rate of elongation on the single-stranded region of the template. Parallel experiments conducted with avian myeloblastosis virus reverse transcriptase showed that this enzyme was not able to unwind the RNA duplex, suggesting that strand displacement by poliovirus 3DP01 is not a property shared by all polymerases.

“…A small, highly polar polypeptide, 3B (also known as VPg), is found covalently linked to all newly synthesized viral plusand minus-strand RNAs and to nascent strands of the replicative intermediate (48). 3B has been proposed to serve as a primer for plus-strand RNA synthesis (46,48,(61)(62)(63). Several observations support this idea.…”

mentioning

confidence: 99%

The antiviral compound enviroxime targets the 3A coding region of rhinovirus and poliovirus

Heinz

Vance

1995

124

Enviroxime is an antiviral compound that inhibits the replication of rhinoviruses and enteroviruses. We have explored the mechanism of action of enviroxime by using poliovirus type 1 and human rhinovirus type 14 as model systems. By varying the time of drug addition to virus-infected cells, we determined that enviroxime could be added several hours postinfection without significant loss of inhibition. This suggested that the drug targeted a step involved in RNA replication or protein processing. To identify this target, we mapped 23 independent mutations in mutants that could multiply in the presence of 1 g of enviroxime per ml. Each of these mutants contained a single nucleotide substitution that altered one amino acid in the 3A coding region. Using oligonucleotide-directed mutagenesis of cDNA clones, we have confirmed that these single-amino-acid substitutions are sufficient to confer the resistance phenotype. In addition, we conducted two experiments to support the hypothesis that enviroxime inhibits a 3A function. First, we determined by dot blot analysis of RNA from poliovirus-infected cells that enviroxime preferentially inhibits synthesis of the viral plus strand. Second, we demonstrated that enviroxime inhibits the initiation of plus-strand RNA synthesis as measured by the addition of [ 32 P]uridine to 3AB in poliovirus crude replication complexes. To our knowledge, this is the first evidence that 3A can be targeted by antiviral drugs. We anticipate that enviroxime will be a useful tool for investigating the natural function of the 3A protein.