No abstract

Lysolecithin permeabilization of vaccinia virus-infected cells was employed to prepare extracts that support faithful transcription initiation in vitro on plasmids possessing early, intermediate, and late viral gene promoters. Conditions which optimize transcription from each promoter were defined. The in vitro system was used to investigate the multifunctional viral mRNA capping enzyme, which also functions as the viral early gene transcription termination factor (VTF) and a viral intermediate gene transcription initiation factor. A low level of signal-dependent termination of early gene transcription was observed in vitro which could be elevated by the addition of pure mRNA capping enzyme. VTF-dependent transcription termination was found to be restricted to templates that possessed an early promoter. This restriction mimics that observed in vivo and demonstrates that transcription termination is limited to RNA polymerase molecules that recognize early rather than intermediate or late gene promoters. Extracts prepared from cells infected at the nonpermissive temperature with a virus containing a ts mutation in gene D12L, which encodes the small subunit of VTF, are incapable of supporting both early gene transcription termination and intermediate gene transcription initiation. Both activities are restored upon addition of the purified wild-type mRNA capping enzyme.

Vaccinia virus genes A18 and G2 affect the elongation and termination of postreplicative viral gene transcription in opposite ways. Viruses with mutations in gene A18 produce abnormally long transcripts, indicating that A18 is a negative transcription elongation factor. Viruses containing mutations in gene G2 produce transcripts that are abnormally short, truncated specifically from their 3' ends, indicating that G2 is a positive transcription elongation factor. Despite the fact that both A18 and G2 are essential genes, A18-G2 double-mutant viruses are viable, presumably because the effects of the mutations are mutually compensatory. In addition, the anti-poxviral drug isatin-beta-thiosemicarbazone (IBT) seems to enhance elongation during a vaccinia infection: IBT treatment of a wildtype vaccinia infection induces a phenotype identical to an A18 mutant infection, and G2 mutant viruses are dependent on IBT for growth, presumably because IBT restores the G2 mutant truncated transcripts to a normal length. These observations inspire two independent genetic selections that have now been used to identify an additional vaccinia gene, J3, that regulates postreplicative transcription elongation. In the first selection, a single virus that contains an extragenic suppressor of the A18 temperature-sensitive mutant, Cts23, was isolated. In the second selection, several spontaneous IBT-dependent (IBT(d)) mutant viruses were isolated and characterized genetically. Marker rescue mapping and DNA sequence analysis show that the extragenic suppressor of Cts23 contains a point mutation in the J3 gene, while each of seven new IBT(d) mutants contains null mutations in the J3 gene. The J3 protein has previously been identified as a (nucleoside-2'-O-)-methyltransferase and as a processivity subunit for the heterodimeric viral poly(A) polymerase. The nature of the two independent selections used to isolate the J3 mutants strongly suggests that the J3 protein serves as a positive postreplicative transcription elongation factor during a normal virus infection.

Vaccinia uses actin-based motility for virion movement in host cells, but the specific protein components have yet to be defined. A cardinal feature of Listeria and Shigella actin-based motility is the involvement of vasodilatorstimulated phosphoprotein (VASP). This essential adapter recognizes and binds to actin-based motility 1 (ABM-1) consensus sequences [(D͞E)FPPPPX(D͞E), X ‫؍‬ P or T] contained in Listeria ActA and in the p90 host-cell vinculin fragment generated by Shigella infection. VASP, in turn, provides the ABM-2 sequences [XPPPPP, X ‫؍‬ G, P, L, S, A] for binding profilin, an actin-regulatory protein that stimulates actin filament assembly. Immunolocalization using rabbit anti-VASP antibody revealed that VASP concentrates behind motile virions in HeLa cells. Profilin was also present in these actin-rich rocket tails, and microinjection of 10 M (intracellular) ABM-2 peptide (GPPPPP) 3 blocked vaccinia actin-based motility. Vinculin did not colocalize with VASP on motile virions and remained in focal adhesion contacts; however, another ABM-1-containing host protein, zyxin, was concentrated at the rear of motile virions. We also examined time-dependent changes in the location of these cytoskeletal proteins during vaccinia infection. VASP and zyxin were redistributed dramatically several hours before the formation of actin rocket tails, concentrating in the viral factories of the perinuclear cytoplasm. Our findings underscore the universal involvement of ABM-1 and ABM-2 docking sites in actinbased motility of Listeria, Shigella, and now vaccinia.The association of actin filaments with intracellular vaccinia was first discovered over two decades ago (1). However, characterization of the mechanisms responsible for virion-induced host-cell actin filament assembly has proved to be a daunting task. One strategy for exploring how vaccinia induces actin assembly is to identify and assess the contribution of viral protein analogues of cytoskeleton components known to be involved in actin-based motility. For example, genetic deletion of vaccinia's A42R openreading frame for a profilin-like protein failed to impair actin assembly (2), and there was no effect on deletion of the F8L viral protein containing a 42 residue region with homology to the Listeria ivanovi surface protein iActA (3). On the other hand, two viral membrane proteins, B5R and A34R, do affect actin rocket tail formation (4-7). Viral maturation and subsequent locomotory competence require that the intracellular mature virion be wrapped by membranes derived from the trans-Golgi network or by early endosomes to form the intracellular enveloped virion (6). Subsequently, the outer membrane of the intracellular enveloped virion fuses with the plasma membrane, releasing an extracellular enveloped virion. B5R and A34R affect intracellular enveloped virion and extracellular enveloped virion formation, respectively (5, 7). The observation that mutants defective in intracellular enveloped virion and extracellular enveloped virion formation are defect...