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Vaccinia virus ( Orthopoxvirus ) F17 protein is a major virion structural phosphoprotein having a molecular weight of 11 kDa. Recently, it was shown that F17 synthesised in infected cells interacts with mTOR subunits to evade cell immunity and stimulate late viral protein synthesis. Several years back, we purified an 11 kDa protein that inhibited protein synthesis in reticulocyte lysate from virions, and that possesses all physico-chemical properties of F17 protein. To investigate this discrepancy, we used defective vaccinia virus particles devoid of the F17 protein (designated iF17 - particles) to assess their ability to inhibit protein synthesis. To this aim, we purified iF17 - particles from cells infected with a vaccinia virus mutant which expresses F17 only in the presence of IPTG. The SDS-PAGE protein profiles of iF17 - particles or derived particles, obtained by solubilisation of the viral membrane, were similar to that of infectious iF17 particles. As expected, the profiles of full iF17 - particles and those lacking the viral membrane were missing the 11 kDa F17 band. The iF17 - particles did attach to cells and injected their viral DNA into the cytoplasm. Co-infection of the non-permissive BSC40 cells with a modified vaccinia Ankara (MVA) virus, expressing an mCherry protein, and iF17 - particles, induced a strong mCherry fluorescence. Altogether, these experiments confirmed that the iF17 - particles can inject their content into cells. We measured the rate of protein synthesis as a function of the multiplicity of infection (MOI), in the presence of puromycin as a label. We showed that iF17 - particles did not inhibit protein synthesis at high MOI, by contrast to the infectious iF17 mutant. Furthermore, the measured efficiency to inhibit protein synthesis by the iF17 mutant virus generated in the presence of IPTG, was threefold to eightfold lower than that of the wild-type WR virus. The iF17 mutant contained about threefold less F17 protein than wild-type WR. Altogether these results strongly suggest that virion-associated F17 protein is essential to mediate a stoichiometric inhibition of protein synthesis, in contrast to the late synthesised F17. It is possible that this discrepancy is due to different phosphorylation states of the free and virion-associated F17 protein.

In this article, we describe the development and evaluation of a double antigen sandwich enzyme-linked immunosorbent assay (ELISA) able to detect serotype 4-specific antibodies from BTV-4 infected or vaccinated animals using a recombinant BTV-4 VP2 protein. The coding sequence of VP2 was inserted into a pVote plasmid by recombination in the Gateway ® cloning system. Vaccinia virus (VacV) was used as a vector for the expression of the recombinant VP2. After production in BSR cells, recombinant VP2 was purified by immunoprecipitation using a FLAG tag and then used both as the coated ELISA antigen and as the HRP-tagged conjugate. The performance of the ELISA was evaluated with 1186 samples collected from BTV negative, infected or vaccinated animals. The specificity and sensitivity of the BTV-4 ELISA were above the expected standards for the detection of anti-BTV-4 VP2 antibodies in animals reared in Europe or in the Mediterranean basin. Cross-reactions were observed with reference sera for serotypes 10 and 20, and to a lesser extent with serotypes 12, 17 and 24, due to their genetic proximity to serotype 4. Nevertheless, these serotypes have never been detected in Europe and the Mediterranean area. This ELISA, which requires only the production of a recombinant protein, can be used to detect BTV serotype 4-specific antibodies and is therefore an attractive alternative diagnostic method to serum neutralization.

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.

The phosphorylation state of H5R protein was investigated by two-dimensional gel electrophoresis of proteins of BSC-40 cells infected at 32 degrees or 39.5 degrees with vaccinia virus ts mutants of the viral B1R or F10L protein kinase genes. A temperature-dependent increase of underphosphorylated H5R protein (pI 6.8) was demonstrated in the case of the B1R, but not of the F10L gene. The temperature-dependent cytoplasmic location of underphosphorylated H5R protein after infection with the ts mutants of the B1R gene was the consequence of the associated viral DNA replication block. These results show that the B1R protein kinase controls the phosphorylation state of the H5R protein synthesised at the early stage in vaccinia-virus-infected cells., (Copyright 2000 S. Karger AG, Basel.)

The thymidine analogue 4-thiothymidine (s4T) strongly absorbs light at wavelengths in the UVA range (lambda max 335 nm) and we have examined the photoinactivation of vaccinia and herpes simplex viruses grown in the presence of this nucleoside. The cells used in this study (Vero, mouse 1D-TK+) were able to grow at the same rate when cultured in the presence of 2 mM s4T or 2 mM thymidine, albeit at a slower rate than control cells. Consistent with this finding, viruses grown in the presence of 1-4 mM s4T were obtained in reduced yield but retained full infectivity. Both viruses were specifically inactivated by irradiation with 365 nm light and their photosensitivity, as measured by the initial slope of the inactivation curve, increased in parallel with the concentration of s4T added to the culture medium. More than 90% of vaccinia virus grown in the presence of 4 mM s4T was inactivated. Organomercurial agarose chromatography of sheared DNA isolated from vaccinia virus grown in the presence of 2 mM s4T showed that approximately 2.5% of DNA fragments were specifically retained, as compared to 0.2% for control DNA. This value corresponds to at least one s4T residue incorporated per 30,000 nucleotides of vaccinia virus DNA. In fact, it is likely that this ratio is actually approximately 10 times higher because of the incomplete retention of control thiolated oligodeoxynucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the production of Gag particles by Vero, CV-1, or 1D cells infected with different vaccinia virus recombinants expressing HIV gag or gag-pol genes. Immunoblots of (centrifuged) culture media from 1D cells infected with vMM5, a vaccinia virus recombinant expressing the HIV-2 gag-pol genes, revealed the presence of abundant particles that contained (mostly processed) Gag antigens. In contrast, Gag particles were found only in low amounts in the culture medium from Vero cells infected with the same HIV gag-pol vaccinia virus recombinant; the Gag precursor remained associated with the infected Vero cells and was efficiently processed. This low excretion of Gag particles after infection of Vero cells with vMM5 was also demonstrated by assays of reverse transcriptase activity in the pellet of centrifuged culture medium. Cell fractionation showed that Gag proteins were predominantly found in the membrane fraction from both 1D and Vero cells. Electron microscopy observations of 1D or of Vero cells infected with vMM5 vaccinia virus recombinant revealed in both cases the presence of particles budding at the plasma membrane. However, the shape of the budding particles was different in the two cell lines, with immature forms present in the membrane from the infected Vero cells. An inefficient excretion of Gag particles was also observed after infection of Vero cells with different vaccinia virus recombinants expressing either an uncleaved HIV-2 Gag protein or the HIV-1 gag-pol genes, as judged both by immunoblot and reverse transcriptase activity assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Infection with vaccinia virus (a poxvirus) quickly and efficiently shuts off host protein synthesis in the presence of actinomycin D (refs 3--5) or cycloheximide. The cellular messenger RNA apparently remains stable in the infected cells exposed to inhibitors of viral gene transcription. In some cases vaccinia viral RNA or poly(A) synthesis have been implicated in the establishment of this effect. However, in the presence of cordycepin (3-deoxyadenosine) which blocks viral gene transcription and cytoplasmic poly(A) synthesis, cellular protein synthesis is still efficiently inhibited in vaccinia virus-infected cells. This shutoff is also observed in vitro, in the corresponding cell-free extracts, and in a reticulocyte lysate. Therefore the shutoff of host protein synthesis is probably mediated by a factor associated with vaccinia virions. We now report that the formation of the 40S--Met-tRNAfMet initiation complex is inhibited in cytoplasmic extracts derived from vaccinia virus-infected cells exposed to cordycepin to block viral gene expression. A similar inhibition is found in reticulocyte lysates incubated with purified vaccinia cores, confirming the hypothesis that the factor associated with the viral cores is responsible for the inhibition observed in vaccinia virus-infected cells exposed to inhibitors of transcription.

Ribosomal protein phosphorylation was investigated in Ehrlich ascites tumor cells infected with vaccinia virus (Copenhagen strain). After 90 min of simultaneous infection and 32P-labelling, ribosomal proteins Sa, S2 and S13 appear specifically phosphorylated as well as Sb/La, P1 and S6, which are also phosphorylated in control cells. Sa is an acidic protein, whose phosphorylation has not been observed previously. A kinetic study showed that S2 is phosphorylated very rapidly within 10 min after the beginning of infection and it is complete 1 h later. The phosphorylation of S13 begins after a lag time of about 1 h and is completed after about 2.5 h of infection. Moreover only one phosphate is incorporated into S13 on a serine residue while up to four phosphates are incorporated into S2, the first on a serine and the three following on threonine residues. In vivo experiments, carried out in the presence of cycloheximide and cordycepin, suggest a viral origin for the kinase involved in the phosphorylation of S2 and S13. Moreover, in vitro experiments demonstrated that the kinase associated with the viral cores is capable of phosphorylating S2 on a serine residue only. In our cell/virus system, no significant difference in S6 phosphorylation was detected, when compared to uninfected cells. It is concluded that the specific and efficient phosphorylation of three ribosomal proteins from the 40S ribosomal subunit correlate well with possible translational mechanisms ensuring the efficient expression of early and late genes of vaccinia virus. In the light of these and previous results [Person, A. and Beaud, G. (1986) J. Biol. Chem. 261, 8283-8289], a mechanism is proposed for the shut-off of host protein synthesis and the selective translation of mRNAs of viral origin.

Translation of cellular and early vaccinia RNA in nuclease-treated lysates, derived from uninfected and vaccinia-virus-infected cells at the early stage, has been investigated. When using limiting amounts of RNA no discrimination of translation was observed in the infected cells lysates; this conclusion was confirmed by sensitive RNA competition experiments for translation in vitro and also when using two different fractionated systems for protein synthesis in vitro. This absence of detectable discrimination in vitro was established both by comparing incorporation of [35S]methionine into proteins and by analysis of the products thus synthesized by sodium dodecylsulfate gel electrophoresis. However, a modification of the translational machinery from vaccinia-virus-infected cells did occur since the only the ribosomal salt wash derived from infected cells was able to reverse the inhibition of protein synthesis in vitro resulting from excess RNA (control or early). This property of vaccinia-virus-infected cell lysates may result from the synthesis l machinery from vaccinia-virus-infected cells did occur since the only the ribosomal salt wash derived from infected cells was able to reverse the inhibition of protein synthesis in vitro resulting from excess RNA (control or early). This property of vaccinia-virus-infected cell lysates may result from the synthesis l machinery from vaccinia-virus-infected cells did occur since the only the ribosomal salt wash derived from infected cells was able to reverse the inhibition of protein synthesis in vitro resulting from excess RNA (control or early). This property of vaccinia-virus-infected cell lysates may result from the synthesis of an early protein involved in translation or from a better recovery of translational factors from the infected cells, as suggested in the accompanying paper.

We have reported the isolation of cis-acting regulatory DNA sequences promoting expression of the herpes virus thymidine kinase gene in vaccinia virus recombinants. In this work we show that each of the inserts from recombinants VpT25, 28, 36 and 56 contains a vaccinia virus early promoter. The position of each of the early RNA start sites in the nucleotide sequence of these four vaccinia virus inserts was precisely mapped by an S1 nuclease mapping procedure. Among the four recombinants analysed only VpT56-infected cells also contained a substantial amount of a transcript with the same 5' end at late period. The insert present in VpT25 contained a new late RNA start site 50 nucleotides upstream from that of the early RNA. The four inserts were mapped on the vaccinia virus genome. We also localized the 5' end of the mRNA of a vaccinia virus host-range gene, whose DNA nucleotide sequence has recently been established. The 45 nucleotides preceding the RNA start site from most of 19 known vaccinia virus early promoters were found to be A + T-rich (at least 80%) and contained shorter A-rich (at least 60%) regions, beginning approximately 25 nucleotides upstream from the RNA start site. The information content, as expressed by the parameter Rsequence, of early vaccinia virus promoters revealed ten bits of information in the sequence of 28 nucleotides upstream from the early RNA start sites. Most of the information needed to locate an early promoter is contained within the nucleotide sequence upstream from an RNA start site. A consensus sequence consists of two blocks: the sequence AA(A/T)N(T/A)N(A/G)AAAANAANA starting at position -27 and the sequence (T/A)(C/T)N(A/T)T(A/G) starting at position -5. It was concluded that vaccinia virus early promoters may be characterized by an A + T-rich region of approximately 45 nucleotides preceding the RNA start site and include a specific 3'-terminal sequence of 28 nucleotides containing at least ten bits of information. A procedure for localizing putative early RNA start sites in nucleotide sequences is proposed.

When vaccinia-virus-infected cells were labeled with radioactive phosphate in the absence of viral gene expression an additional phosphoprotein, containing phosphoserine, was found specifically associated with the ribosomes. The phosphoprotein was removed from the ribosomes following a 0.5 M KCl washing or after EDTA treatment. This additional phosphoprotein was found in infected cells after either a long (3-4 h) or a short (30 min) labeling period; it was detected when the infected cells were incubated in the presence or absence of an inhibitor of RNA or protein synthesis. This phosphoprotein originated from the phosphorylation of vaccinia virion structural protein VP11b (Mr 11,000) at a specific site since only a single major phosphopeptide was obtained after trypsin digestion. This phosphoprotein was also present in purified vaccinia virions labeled with radioactive phosphate. VP11b protein was phosphorylated in vitro by the protein kinase associated with the cores. When the reaction was carried out at an alkaline pH the phosphorylation in vitro occurred at different sites in the protein; at neutral pH the phosphorylation of VP11b was more specific and, as judged by tryptic peptide analysis, occurred mainly at the same site as in the phosphorylation in vivo. A role for the involvement of phosphoprotein VP11b in the establishment of the shut off of host protein synthesis by vaccinia virus is suggested.

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 same mono-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.

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.

DNA fragments preceding open reading frames in a conserved segment of the vaccinia virus genome (Plucienniczak A., et al. (1985) Nucleic Acids Res. 13, 985-998) were cloned into plasmids upstream of the S gene of the hepatitis B virus encoding the surface antigen (HBsAg). Recombinant vaccinia virus obtained after insertion of these constructs into the thymidine kinase gene were used to infect mouse 1D cells. HBsAg was assayed in cellular supernatants. A strong promoter was thus identified in a 295 bp fragment preceding the coding region of the 147 kDa subunit of the vaccinia RNA polymerase.

A method for preparing detergent cytoskeletons from uninfected or vaccinia-virus-infected cells is described. This method resulted in the fractionation of the cytoplasmic compartment into soluble and cytoskeletal fractions. More than 85% of small molecules and tRNA were released from the cytoskeleton and recovered into the soluble fraction. The cytoskeletal fraction contained about 40% of the cytoplasmic proteins and 67% of total cytoplasmic RNA. Similar values were obtained for vaccinia-virus-infected cells. In contrast, whereas 85% of the cellular polysomes and poly(A)-rich RNA remained associated with the cytoskeleton in uninfected cells, more than 40% of vaccinia early mRNA engaged into polysomes was found to be not associated with the cytoskeleton. A similar partition was found when the viral RNA was labeled for 15 min at 5 min and 20 min after the infection or when varying the duration of the pulse. Soluble and cytoskeletal viral polysomes were found to synthesize a similar set of proteins after translation in vitro of the corresponding mRNA. The fate of rapidly labeled cellular poly(A)-rich RNA upon vaccinia virus infection was followed by a glucosamine/uridine chase procedure, and also that of relatively stable poly(A)-rich RNA after long-term labeling and chase. In both cases no release of poly(A)-rich RNA from the cytoskeleton occurred after vaccinia virus infection. These experiments reveal that cellular mRNA remains associated to the cytoskeleton in EAT cells infected with vaccinia virus (early period) whereas at least 40% of the vaccinia early polysomes are not associated with the cytoskeleton. A model for vaccinia early mRNA metabolism is presented, which may account for the rapid shut-off of host protein synthesis.