The Vaccinia Virus Bifunctional Gene J3 (Nucleoside-2′-O-)-methyltransferase and Poly(A) Polymerase Stimulatory Factor Is Implicated as a Positive Transcription Elongation Factor by Two Genetic Approaches

AbstractVaccinia 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-β-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 (IBTd) 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 IBTd 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

The E6 protein from vaccinia virus is required for the formation of immature virions.

An IPTG-inducible mutant in the E6R gene of vaccinia virus was used to study the role of the E6 virion core protein in viral replication. In the absence of the inducer, the mutant exhibited a normal pattern DNA replication, concatemer resolution and late gene expression, but it showed an inhibition of virion structural protein processing it failed to produce infectious particles. Electron microscopic analysis showed that in the absence of IPTG viral morphogenesis was arrested before IV formation: crescents, aberrant or empty IV-like structures, and large aggregated virosomes were observed throughout the cytoplasm. The addition of IPTG to release a 12-h block showed that virus infectious particles could be formed in the absence of de novo DNA synthesis. Our observations show that in the absence of E6 the association of viroplasm with viral membrane crescents is impaired

Phenotypic analysis of a temperature sensitive mutant in the large subunit of the vaccinia virus mRNA capping enzyme

AbstractThe heterodimeric vaccinia virus mRNA capping enzyme is a multifunctional enzyme, encoded by genes D1R and D12L. Published biochemical experiments demonstrate that, in addition to mRNA capping, the enzyme is involved in early viral gene transcription termination and intermediate viral gene transcription initiation. This paper presents the phenotypic characterization of Dts36, a temperature sensitive mutant in the large subunit of the mRNA capping enzyme (G705D), encoded by gene D1R. At the non-permissive temperature, Dts36 displays decreased steady state levels of some early RNAs, suggesting a defect in mRNA capping. Mutant infections also show decreased steady state levels of some early proteins, while DNA replication and post-replicative gene expression are absent. Under non-permissive conditions, the mutant directs synthesis of longer-than-normal early mRNAs from some genes, demonstrating that early gene transcription termination is defective. If mutant infections are initiated at the permissive temperature and shifted to the non-permissive temperature late during infection, steady state levels of intermediate gene transcripts decrease while the levels of late gene transcripts remain constant, consistent with a defect in intermediate gene transcription initiation. In addition to its previously described role in mRNA capping, the results presented in this study provide the first in vivo evidence that the vaccinia virus mRNA capping enzyme plays a role in early gene transcription termination and intermediate gene transcription

Mutation of Vaccinia Virus Gene G2R Causes Suppression of Gene A18R ts Mutants: Implications for Control of Transcription

AbstractThis report provides genetic evidence that two vaccinia virus genes, A18R and G2R, both of which affect the fidelity of viral transcriptionin vivo,interact with each other or act on a common biochemical pathway. Previous experiments with the antipoxviral drug isatin-β-thiosemicarbazone suggest that lethal mutation of gene G2R would compensate for mutations in gene A18R. We therefore tested the hypothesis that gene G2R is an extragenic suppressor of A18R mutations. First, we constructed a recombinant which contains both a G2R deletion mutation and an A18R temperature-sensitive mutation and found that this recombinant was viable. Second, we isolated both cold-sensitive and temperature-insensitive phenotypic revertants of A18R temperature-sensitive mutants and found in both cases that the revertants contained G2R mutations. In the case of the cold-sensitive revertants, we were able to prove that the cold-sensitive phenotype mapped to the G2R gene. Combined with the biochemical data on A18R and G2R, these results imply that the A18R and G2R genes interact with each other either directly or indirectly in a fashion which affects the fidelity of intermediate and late viral transcription

Vaccinia virus temperature-sensitive mutants in the A28 gene produce non-infectious virions that bind to cells but are defective in entry

AbstractThe vaccinia virus temperature-sensitive mutations Cts6 and Cts9 were mapped by marker rescue and DNA sequencing to the A28 gene. Cts6 and Cts9 contain an identical 2-bp deletion truncating the A28 protein and removing the fourth conserved cysteine near the C-terminus. Cts9 mutant virions produced at 40 °C were non-infectious and unable to cause cytopathic effect. However, the mutant A28 protein localized to purified mature virions (MV) at 31 °C and 40 °C. MV of Cts9 produced at 40 °C bound to cells but did not enter cells. Low pH treatment of Cts9-infected cells at 18 h p.i. failed to produce fusion from within at 40 °C, but gave fusion at 31 °C. Adsorption of Cts9 mutant virions to cells followed by low pH treatment showed a defect in fusion from without. The Cts9 phenotype suggests that the A28 protein is involved in both virus entry and cell–cell fusion, and supports the linkage between the two processes

Temperature-sensitive mutant in the vaccinia virus E6 protein produce virions that are transcriptionally inactive.

The vaccinia virus E6R gene encodes a late protein that is packaged into virion cores. A temperature-sensitive mutant was used to study the role of this protein in viral replicative cycle. Cts52 has a P226L missense mutation in the E6R gene, shows a two-log reduction in plaque formation, but displays normal patterns of gene expression, late protein processing and DNA replication during infection. Mutant virions produced at 40 degrees C were similar in their morphology to wt virions grown at 40 degrees C. The particle to infectivity ratio was 50 times higher in purified Cts52 grown at 40 degrees C when compared to the mutant grown at permissive temperature. In vitro characterization of Cts-52 particles grown at 40 degrees C revealed no differences in protein composition or in DNA content and the mutant virions could bind and enter cells. However, core particles prepared from Cts52 grown at 40 degrees C failed to transcribe in vitro. Our results show that E6 in the virion has either a direct or an indirect role in viral transcription

Interaction between the J3R Subunit of Vaccinia Virus Poly(A) Polymerase and the H4L Subunit of the Viral RNA Polymerase

AbstractJ3R, the 39-kDa subunit of vaccinia virus poly(A) polymerase, is a multifunctional protein that catalyzes (nucleoside-2′-O-)-methyltransferase activity, serves as a poly(A) polymerase stimulatory factor, and acts as a postreplicative positive transcription elongation factor. Prior results support an association between poly(A) polymerase and the virion RNA polymerase. A possible direct interaction between J3R and H4L subunit of virion RNA polymerase was evaluated. J3R was shown to specifically bind to H4L amino acids 235–256, C terminal to NPH I binding site on H4L. H4L binds to the C-terminal region of J3R between amino acids 169 and 333. The presence of a J3R binding site near to the NPH I binding region on H4L led us to evaluate a physical interaction between NPH I and J3R. The NPH I binding site was located on J3R between amino acids 169 and 249, and J3R was shown to bind to NPH I between amino acids 457 and 524. To evaluate a role for J3R in early gene mRNA synthesis, transcription termination, and/or release, a transcription-competent extract prepared from cells infected with mutant virus lacking J3R, J3-7. Analysis of transcription activity demonstrated that J3R is not required for early mRNA synthesis and is not an essential factor in early gene transcription termination or transcript release in vitro. J3R interaction with NPH I and H4L may serve as a docking site for J3R on the virion RNA polymerase, linking transcription to mRNA cap formation and poly(A) addition

Use of Lysolecithin-Permeabilized Infected-Cell Extracts to Investigate thein VitroBiochemical Phenotypes of Poxvirus ts Mutations Altered in Viral Transcription Activity

AbstractLysolecithin permeabilization of vaccinia virus-infected cells was employed to prepare extracts that support faithful transcription initiationin vitroon plasmids possessing early, intermediate, and late viral gene promoters. Conditions which optimize transcription from each promoter were defined. Thein vitrosystem 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 observedin vitrowhich 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 observedin vivoand 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

Functional Traits of Tropical Trees and Lianas Explain Spatial Structure across Multiple Scales

Dispersal and density dependence are major determinants of spatial structure, population dynamics and coexistence for tropical forest plants. However, because these two processes can jointly influence spatial structure at similar scales, analysing spatial patterns to separate and quantify them is often difficult. Species functional traits can be useful indicators of dispersal and density dependence. However, few methods exist for linking functional traits to quantitative estimates of these processes that can be compared across multiple species. We analysed static spatial patterns of woody plant populations in the 50 ha Forest Dynamics Plot on Barro Colorado Island, Panama with methods that distinguished scale‐specific differences in species aggregation. We then tested how these differences related to seven functional traits: growth form, dispersal syndrome, tree canopy layer, adult stature, seed mass, wood density and shade tolerance. Next, we fit analytically tractable spatial moment models to the observed spatial structure of species characterized by similar trait values, which allowed us to estimate relationships of functional traits with the spatial scale of dispersal, and the spatial scale and intensity of negative density dependence. Our results confirm that lianas are more aggregated than trees, and exhibit increased aggregation within canopy gaps. For trees, increased seed mass, wood density and shade tolerance were associated with less intense negative density dependence, while higher canopy layers and increased stature were associated with decreased aggregation and better dispersal. Spatial structure for trees was also strongly determined by dispersal syndrome. Averaged across all spatial scales, zoochory was more effective than wind dispersal, which was more effective than explosive dispersal. However, at intermediate scales, zoochory was associated with more aggregation than wind dispersal, potentially because of differences in short‐distance dispersal and the intensity of negative density dependence. Synthesis. We develop new tools for identifying significant associations between functional traits and spatial structure, and for linking these associations to quantitative estimates of dispersal scale and the strength and scale of density dependence. Our results help clarify how these processes influence woody plant species on Barro Colorado, and demonstrate how these tools can be applied to other sites and systems