Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants

The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest

Expression of Sindbis virus structural proteins via recombinant vaccinia virus: synthesis, processing, and incorporation into mature Sindbis virions

We have obtained a vaccinia virus recombinant which contains a complete cDNA copy of the 26S RNA of Sindbis virus within the thymidine kinase gene of the vaccinia virus genome. This recombinant constitutively transcribed the Sindbis sequences throughout the infectious cycle, reflecting the dual early-late vaccinia promoter used in this construction. The Sindbis-derived transcripts were translationally active, giving rise to both precursor and mature structural proteins of Sindbis virus, including the capsid protein (C), the precursor of glycoprotein E2 (PE2), and the two mature envelope glycoproteins (E1 and E2). These are the same products translated from the 26S mRNA during Sindbis infection, and thus these proteins were apparently cleaved, glycosylated, and transported in a manner analogous to that seen during authentic Sindbis infections. By using epitope-specific antibodies, it was possible to demonstrate that recombinant-derived proteins were incorporated into Sindbis virions during coinfections with monoclonal antibody-resistant Sindbis variants. These results suggest that all the information necessary to specify the proper biogenesis of Sindbis virus structural proteins resides within the 26S sequences and that vaccinia may provide an appropriate system for using DNA molecular genetic manipulations to unravel a variety of questions pertinent to RNA virus replication

A host cell membrane protein, golgin-97, is essential for poxvirus morphogenesis

AbstractAcquisition of the membrane and genome encapsidation is an important step in the replication of enveloped viruses. The biogenesis of the poxviral primary membrane and the core as well as the mechanisms of their maturation are poorly understood. Using RNA interference approach, we demonstrate that a cellular trans-Golgi network membrane protein, golgin-97, is essential for virus replication. Analysis of the virion morphology in the cells depleted of golgin-97 shows that the protein is required for the virus morphogenesis and, in particular, for the formation of the first infectious virus form, mature virus, but not its precursor, immature virus. This suggests that golgin-97 may be involved in the maturation of the virus core and, potentially, the virus membrane

A conditional-lethal vaccinia virus mutant demonstrates that the I7L gene product is required for virion morphogenesis

A conditional-lethal recombinant virus was constructed in which the expression of the vaccinia virus I7L gene is under the control of the tetracycline operator/repressor system. In the absence of I7L expression, processing of the major VV core proteins is inhibited and electron microscopy reveals defects in virion morphogenesis subsequent to the formation of immature virion particles but prior to core condensation. Plasmid-borne I7L is capable of rescuing the growth of this virus and rescue is optimal when the I7L gene is expressed using the authentic I7L promoter. Taken together, these data suggest that correct temporal expression of the VV I7L cysteine proteinase is required for core protein maturation, virion assembly and production of infectious progeny

Development of an in vitro cleavage assay system to examine vaccinia virus I7L cysteine proteinase activity

Through the use of transient expression assays and directed genetics, the vaccinia virus (VV) I7L gene product has been implicated as the major maturational proteinase required for viral core protein cleavage to occur during virion assembly. To confirm this hypothesis and to enable a biochemical examination of the I7L cysteine proteinase, an in vitro cleavage assay was developed. Using extracts of VV infected cells as the source of enzyme, reaction conditions were developed which allowed accurate and efficient cleavage of exogenously added core protein precursors (P4a, P4b and P25K). The cleavage reaction proceeded in a time-dependent manner and was optimal when incubated at 25°C. I7L-mediated cleavage was not affected by selected inhibitors of metalloproteinases, aspartic acid proteinases or serine proteinases (EDTA, pepstatin, and PMSF, respectively), but was sensitive to several general cysteine proteinase inhibitors (E-64, EST, Iodoacetic acid, and NEM) as well as the I7L active site inhibitor TTP-6171 [C. Byrd et al., J. Virol. 78:12147–12156 (2004)]. Finally, in antibody pull down experiments, it could be demonstrated that monospecific αI7L serum depleted the enzyme activity whereas control sera including αG1L, directed against the VV metalloproteinase, did not. Taken together, these data provide biochemical evidence that I7L is a cysteine proteinase which is directly involved in VV core protein cleavage. Furthermore, establishment of this I7L-mediated in vitro cleavage assay should enable future studies into the enzymology and co-factor requirements of the proteolysis reaction, and facilitate antiviral drug development against this essential target

Importance of disulphide bonds for vaccinia virus L1R protein function

L1R, a myristylated late gene product of vaccinia virus, is essential for formation of infectious intracellular mature virions (IMV). In its absence, only viral particles arrested at an immature stage are detected and no infectious progeny virus is produced. Previous studies have shown that the L1R protein is exclusively associated with the IMV membrane and that myristylation is required for correct targeting. The L1R protein contains six cysteine amino acid residues that have all been shown to participate in intramolecular disulphide bonds. However, it was not clear what role, if any, the disulfide bonds play in the membrane topology of the L1R protein. To address this question, a comprehensive library of L1R mutants in which the cysteine residues have been mutated to serine (either individually or in combination) were tested for their ability to rescue a L1R conditional lethal mutant virus under non-permissive conditions. Much to our surprise, we determined that C57 was not essential for production of infectious IMV. These results suggest that protein disulphide isomerases may be involved in reorganization of disulfide bonds within the L1R protein

Mutational analysis of the potential catalytic residues of the VV G1L metalloproteinase

The vaccinia virus G1L open-reading frame is predicted to be a metalloproteinase based upon the presence of a conserved zinc-binding motif. Western blot analysis demonstrates G1L undergoes proteolytic processing during the course of infection, although the significance of this event is unknown. In order to determine which amino acid residues are important for G1L activity, a plasmid-borne library of G1L constructs containing mutations in and about the active site was created. Transient expression analysis coupled with a trans complementation assay of a conditionally-lethal mutant virus suggest that, of the mutants, only glutamic acid 120 is non-essential for G1L processing to occur

Analysis of vaccinia virus temperature-sensitive I7L mutants reveals two potential functional domains

As an approach to initiating a structure-function analysis of the vaccinia virus I7L core protein proteinase, a collection of conditional-lethal mutants in which the mutation had been mapped to the I7L locus were subjected to genomic sequencing and phenotypic analyses. Mutations in six vaccinia virus I7L temperature sensitive mutants fall into two groups: changes at three positions at the N-terminal end between amino acids 29 and 37 and two different substitutions at amino acid 344, near the catalytic domain. Regardless of the position of the mutation, mutants at the non-permissive temperature failed to cleave core protein precursors and had their development arrested prior to core condensation. Thus it appears that the two clusters of mutations may affect two different functional domains required for proteinase activity

NE Iowa Watershed and Karst Map

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