Georges Beaud

The punctate sites of accumulation of vaccinia virus early proteins are precursors of sites of viral DNA synthesis.

Several vaccinia virus early proteins (encoded by genes B1R, H5R and I3L) synthesized in the presence of an inhibitor of DNA synthesis localize, at least in part, to punctate inclusions that are visible by immunofluorescence in the cytoplasm of poxvirus-infected cells. It is shown that these inclusions contain DNA (visualized by DAPI staining of the infected cells) and that the number of inclusions is proportional to the amount of input virus. Their mean diameter (about 680 nm) was larger than that of purified vaccinia virus particles. When the inhibition of DNA synthesis was reversed, incorporation of BrdU into the B1R particles was demonstrated after labelling for 30 min, suggesting that these cytoplasmic focal sites correspond to viral DNA replication complexes that have initiated normally but are inhibited at the step of DNA chain elongation. These experiments suggest strongly that these inclusions are the precursors of the virosomes.

Preferential virosomal location of underphosphorylated H5R protein synthesized in vaccinia virus-infected cells.

The phosphorylation state of vaccinia virus (VV) protein H5R synthesized in infected cells was investigated by two-dimensional gel electrophoresis. Most of the H5R protein was underphosphorylated (pI 5.9 to 6.8) and, on centrifugation of cell lysates, was associated with virosomes sedimenting with nuclei. However, about a quarter of the H5R protein synthesized was highly phosphorylated (pI 5.5), and this was the major form of the H5R protein present in cytoplasmic extracts. Immunofluorescence of VV-infected cells in the absence of DNA replication showed that underphosphorylated H5R protein, specifically recognized by antibody, was abundantly distributed throughout the cytoplasm but also present in punctate particles, whereas most of the B1R protein detected was in the punctate particles. Late gene expression was not required for the H5R protein to accumulate in virosomes--viral DNA synthesis was sufficient. The different phosphorylation states and cytological locations of the H5R protein suggest it has multiple roles in VV development.

Ribosomal protein S2/Sa kinase purified from HeLa cells infected with vaccinia virus corresponds to the B1R protein kinase and phosphorylates in vitro the viral ssDNA-binding protein

A ribosomal protein S2 kinase was purified 6000-fold from cytoplasmic extracts of HeLa cells infected with vaccinia virus, using 80S ribosomes or 40S ribosomal subunits as a substrate. Although the preparation was not homogeneous, a 34K component was identified, the chromatographic behaviour of which correlated with enzyme activity. During its purification the ribosomal protein S2 kinase was resolved from a less abundant ribosomal protein S13 kinase, demonstrating the two to be different entities. A second protein kinase activity against a 43K ribosomal protein comigrated with the ribosomal protein S2 kinase activity during all five chromatographic procedures employed, and we conclude that the two activities are properties of a single species. Two-dimensional gel electrophoresis demonstrated that this second substrate was the acidic ribosomal protein Sa, of isoelectric point approximately 5.2, previously shown to be phosphorylated during infection with vaccinia virus. Another substrate for the ribosomal protein S2/Sa kinase in vitro was the 36K viral ssDNA-binding protein, of isoelectric point approximately 5.0, which is also known to be phosphorylated in vivo. The 34K protein correlating with the catalytic activity in the most purified preparations of the ribosomal protein S2/Sa kinase was recognized by an antibody specific for a protein expressed in Escherichia coli from vaccinia virus gene B1R. This and other evidence suggest strongly that the ribosomal protein S2/Sa kinase is the product of this gene.

Protein synthesis in vaccinia virus-infected cells in the presence of amino acid analogs: A translational control mechanism

Abstract A rapid and efficient block of protein synthesis occurs in EAT or L cells exposed to the amino acid analogs ( p -fluorophenylalanine and canavanine) and further infected by vaccinia virus. Cells preinfected for 30 min and then exposed to the analogs do not show this inhibition of protein synthesis which appears to result from a factor associated with the input virions. It is shown that this translational block occurs also on viral early messenger RNAs since they appear to be normally transcribed in the presence of the analogs, as judged by the incorporation of labeled uridine into total cytoplasmic RNA, the size of the poly(A) plus fraction and its capacity to be translated in vitro . The absence of inhibition of viral messenger RNA translation in the preinfected cells further exposed to the amino acid analogs is likely to result from the rapid synthesis of some early protein either involved in a cascade mechanism of translation or releasing the effect of the inhibitory factor associated with the input vaccinia virions. Separation by two-dimensional gel electrophoresis revealed that most of the viral proteins analyzed were not synthesized according a cascade mechanism of translation. The synthesis of one to two viral proteins may occur through the latter mechanism or alternatively these proteins may be modified after their translation by some virus coded enzyme.

Identification of sites phosphorylated by the vaccinia virus B1R kinase in viral protein H5R

BackgroundVaccinia virus gene B1R encodes a serine/threonine protein kinase. In vitro this protein kinase phosphorylates ribosomal proteins Sa and S2 and vaccinia virus protein H5R, proteins that become phosphorylated during infection. Nothing is known about the sites phosphorylated on these proteins or the general substrate specificity of the kinase. The work described is the first to address these questions.ResultsVaccinia virus protein H5R was phosphorylated by the B1R protein kinase in vitro, digested with V8 protease, and phosphopeptides separated by HPLC. The N-terminal sequence of one radioactively labelled phosphopeptide was determined and found to correspond to residues 81-87 of the protein, with Thr-84 and Thr-85 being phosphorylated. A synthetic peptide based on this region of the protein was shown to be a substrate for the B1R protein kinase, and the extent of phosphorylation was substantially decreased if either Thr residue was replaced by an Ala.ConclusionsWe have identified the first phosphorylation site for the vaccinia virus B1R protein kinase. This gives important information about the substrate-specificity of the enzyme, which differs from that of other known protein kinases. It remains to be seen whether the same site is phosphorylated in vivo.

Transcripts containing a small anti-HIV hammerhead ribozyme that are active in the cell cytoplasm but inactive in vitro as free RNAs.

In order to study the activity of a hammerhead ribozyme in a cytoplasmic environment. HeLa cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase were contransfected with plasmids expressing the ribozyme and its target RNA (nucleotides (nt) +1 to +692 of HIV-1 RNA) under the control of a T7 promoter. Two ribozyme-containing plasmids were designed to express RNAs of respectively 181 nt (Rz181) and 132 nt (Rz132). The sequence of each of these RNAs contained a 35 nt hammerhead ribozyme which is known to cleave its minimal 14-mer RNA substrate efficiently in vitro at a site corresponding to position +115 of the HIV-1 RNA. Control transfections were carried out with the parental plasmid pET3, which expressed a 134 nt RNA lacking the ribozyme sequence, and also with a plasmid expressing a 181 nt RNA (Rz181M) containing a single mutation known to inactivate the in vitro cleavage activity of the ribozyme. As detected by RT-PCR, the amount of target RNA was reproducibly reduced at a ribozyme/target ratio higher than 50 with Rz181 and Rz132 whereas it remained unaffected with Rz181M, thus eliminating the possibility of antisense inhibition. Rz132 proved to be more efficient than Rz181. Competitive RT-PCR indicated that, at ribozyme/target ratio of 300, the amount of residual target RNA was reduced by approximately 85% in the presence of Rz181. In contrast to these in vivo effects, Rz181 and Rz132 obtained by in vitro transcription were inactive against the minimal 14 mer (or longer) substrate under a variety of conditions. In conclusion, although in vitro studies of ribozymes are essential to learn their catalytic mechanism, they cannot be used to predict the efficiency of RNAs containing a ribozyme sequence when it is expressed in cells.

Translation in micrococcal nuclease-treated cell-free extracts from Ehrlich ascites tumor cells: Stimulation by initiation factor eIF-2B

Translation of exogenous mRNAs in micrococcal nuclease-treated extracts from Ehrlich ascites tumor cells is greatly stimulated by the addition of crude initiation factors or initiation factors eIF-2B and eIF-2 containing eIF-2B. The requirement for exogenous eIF-2B in micrococcal nuclease-treated extracts does not result from either loss or enhanced phosphorylation of eIF-2 during incubation.

Identification of induced protein kinase activities specific for the ribosomal proteins uniquely phosphorylated during infection of HeLa cells with vaccinia virus

We have examined the ribosomal protein kinase activities in partially purified cytoplasmic extracts from HeLa cells infected with vaccinia virus. We found an activity or activities, absent from mock‐infected cells, that was capable of phosphorylating the proteins S2 and S13 in vitro. The ribosomes phosphorylated in vitro exhibited the same multiple phosphorylation of S2 found in vivo, at least 3 phosphoryl residues being seen, and the samemono‐phosphorylation of S13. Also as in vivo, ribosomal protein S2 contained phosphothreonine as well as phosphoserine, whereas S13 contained only phosphoserine. This strongly suggests that these new protein kinase activities are responsible for the ribosomal protein phosphorylations that occur during infection with vaccinia virus.

Mapping of a mutation site in the thymidine kinase gene of vaccinia virus by marker rescue

Summary The thymidine kinase (TK)-deficient mutant 1004B of vaccinia virus derived from the IHD strain was rescued with intact genomic DNA obtained from the wild-type IHD or Copenhagen strain of the virus. The mutant was also rescued by a plasmid (pBR-J) composed of pBR322 and the Hin dIII-J genomic fragment (cloned from the WR strain) which contains the wild type TK gene. pBR-J was cleaved with eight restriction enzymes, some of which also cleaved the pBR322 portion of the plasmid, and the digestion products were used to rescue the TK − virus mutant. Cleaving pBR-J with Eco RI reduced the amount of rescued virus by 92%. Further-more, the plot of the yields of rescued virus as a function of the position of enzyme cleavage sites revealed a minimal value and mapped a zero rescue value at about 0.6 kbp. This mapping of a mutation site in the 1004B TK − mutant was relatively precise (within 0.35 kbp), with no mapping of corresponding mRNA. Moreover, the length of the DNA involved in each recombination event for marker rescue with this cloned DNA did not exceed about 2 kbp.

The inhibition of vaccinia virus replication by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB): an effect at the assembly stage.

The step sensitive to DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) in vaccinia virus replication has been investigated. Ninety microM-DRB extensively inhibited the yield of vaccinia virus after infection of Ehrlich ascites tumour cells. DRB did not inhibit cytoplasmic vaccinia DNA replication. Cytoplasmic viral RNA synthesis (both early and late) was also apparently unaffected and the virus RNAs thus synthesized were normal sized. The expression of early, intermediate and late proteins was not detectably impaired by DRB in vaccinia virus-infected cells. DRB inhibited vaccinia virus replication at the assembly stage since most of the virus DNA remained in a DNase-sensitive form in the infected cells and the virus was therefore not normally coated with virus proteins.