Isolation and Characterization of Vesicular Stomatitis Virus PolR Revertants: Polymerase Readthrough of the Leader–N Gene Junction Is Linked to an ATP-Dependent Function

Abstract

The switch from transcription to replication of the VSV genome is coupled to assembly of nascent chains and involves an unspecified change in the P-L polymerase complex when it reaches the leader-N gene junction. PoIR VSV mutants, in contrast to wild-type virus, read through this first gene junction at high frequency without concurrent assembly, and they show altered ATP requirements for transcription in vitro. The mutation(s) responsible for the poIR phenotype segregates to the N-RNA template fraction. We report here that both poIR1 and poIR2 mutants display severe growth restriction in mouse L cells but not in BHK cells. Four of six poIR1 revertant viruses, originating from rare plaques on L cells, showed wild-type characteristics for growth, readthrough of leader-N gene junction, and ATP utilization, while two showed partial and quantitatively parallel coreversion of all properties. Sequence analysis of N and P genes of poIR mutants and revertants provided proof that a single mutation in the N protein, Arg179 to His, is responsible for the poIR phenotype. PoIR1, but not poIR2, also displayed a phenotypically silent GA-to-GG change in the N-P intergenic dinucleotide sequence Five of six revertants retained the poIR1 N protein mutation and showed no change in their P gene. We conclude that the L protein likely contains second-site suppressors of the poIR phenotype, and we propose that the switch from transcription to replication is modulated by an ATP-dependent interaction between the template-associated N protein and the L subunit of the P L polymerase complex.

Functional Analysis of the Genomic and Antigenomic Promoters of Human Respiratory Syncytial Virus

“…If this model is correct, transcription and replication could be mediated by two separate pools of polymerase which have different conformations allowing them to bind the common promoter element at the 3Ј terminus, but then recognize different initiation sites. This is consistent with evidence that VSV transcription and replication are mediated by two subsets of polymerase, differentiated by posttranslational modification (8,31).…”

Cell-Type-Specific Growth Restriction of Vesicular Stomatitis Virus polR Mutants Is Linked to Defective Viral Polymerase Function

“…BHK-21 and L-929 cells, as well as rat fibroblast 3Y1 cells (obtained from Bartholomew Sefton, Salk Institute), were grown as monolayers in Eagle's minimal essential medium (MEM) containing 7% newborn calf serum. Recombinant wt and polR VSV (see below) were grown and titers were determined by plaque assay on BHK cells as described previously (9). Infections were carried out in cell monolayers at 95 to 100% confluence in 5-cm dishes at an effective MOI of 10 PFU/cell unless otherwise stated.…”

“…The presence of this mutation in the assembled virus templates appears to be solely responsible for the observed alterations in viral RNA synthesis and ATP requirements (9,20,32). The properties of polR mutants suggest that the N protein component of assembled viral templates plays a role in determining polymerase function, but the mechanism involved remains unclear.…”

“…polR mutants have since proved very useful in deciphering virus polymerase function, notably, what distinguishes transcriptase from replicase activity and identifying template start sites for the two modes of synthesis (6,9,10,20,33). VSV RNA synthesis takes place entirely in the cytoplasm of infected cells and requires templates fully encapsidated by the viral nucleoprotein N (for reviews, see references 40 and 53).…”

“…Interestingly, the VSV polR mutants display a host range phenotype for growth in some established cell lines (9). The mutants grow nearly as well as wt virus in BHK cells but show very strong growth restriction in mouse L-929 cells.…”