Protocadherin 15 (PCDH15): a new secreted isoform and a potential marker for NK/T cell lymphomas.

Natural killer cells are well known to play an important role in immune defense against tumor development and viral infections. To further characterize new functionally relevant structures in these cells, we studied a series of monoclonal antibodies that we have raised against the NK cell line YT. One of these antibodies previously described as AY19, recognizes a 85 kD surface glycoprotein. Here we report the identification of a new secreted isoform of protocadherin 15, PCDH15C, which represents a potential associated protein for p85. Importantly, whereas protocadherins are absent from the surface of normal hematopoietic cells, we describe, for the first time, that PCDH15 is expressed in cytotoxic tumor-derived T- and NK-cell lines as well as in biopsies of nasal NK/T-cell lymphomas.

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 vaccinia promoters by heterologous expression of hepatitis B surface antigen in mouse cells infected by recombinant vaccinia viruses.

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.

Characterization of vaccinia virus early promoters and evaluation of their informational content.

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.

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 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.

Vaccinia virus DNA replication: a short review.

The purpose of this review is to summarize information published since 1990 on DNA replication, recombination and repair of vaccinia virus, a poxvirus. Temperature-sensitive mutations reveal four essential genes related to viral DNA replication: the E9L DNA polymerase, B1R protein kinase, D5R protein, and D4R uracil DNA glycosylase. Other proteins are likely to be also involved in viral DNA replication: the H6R DNA topoisomerase, I3L single stranded-DNA binding protein, H5R virosome-associated protein, and A50R DNA ligase. In addition, several viral-encoded proteins do regulate the level of the deoxyribonucleoside triphosphate pool: the J2R thymidine kinase, A48R thymidylate kinase, 14L and F4L subunits of ribonucleotide reductase, and F2L dUTPase. Despite the apparent simplicity of the mechanism of vaccinia virus DNA replication, several important questions related to the three Rs remain unsolved.

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.

Identification by mass spectroscopy of three major early proteins associated with virosomes in vaccinia virus-infected cells.

Virosomes are cytoplasmic sites of replication of vaccinia virus DNA and were prepared from virus-infected HeLa cells. The early virosomal proteins were 35S-labelled and SDS polyacrylamide gel electrophoresis revealed the presence of three major early 35S-labelled proteins of 34, 24 and 45 kDa. The masses of molecules present in the 34 and 24 kDa proteins were measured by the convenient and sensitive MALDI TOF mass spectroscopy technique. Identification of the three virosomal proteins was carried out by MALDI mass spectroscopy of corresponding tryptic digests. For each protein at least 13 measured masses matched, within less than 0.1 Da, calculated tryptic peptides of the vaccinia virus proteins H5R (34 kDa), E3L (24 kDa) and E5R (45 kDa). In addition, virosomes contained several structural proteins from the infecting virus and a 45 kDa keratin-related protein. This work demonstrates directly that the abundant early vaccinia virus proteins H5R, E3L and E5R are associated with the virosomes.

Cell line-dependent release of HIV-like gag particles after infection of mammalian cells with recombinant vaccinia viruses.

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)

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

BACKGROUND: Vaccinia 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. RESULTS: Vaccinia 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. CONCLUSIONS: We 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.

Photoinactivation (365 nm) of vaccinia and herpes simplex viruses induced by a new built-in DNA photosensitizer: 4-thiothymidine.

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)

Temperature-dependent phosphorylation state of the H5R protein synthesised at the early stage of infection in cells infected with vaccinia virus ts mutants of the B1R and F10L protein kinases.

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.

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.

Phosphorylation in vivo of a vaccinia-virus structural protein found associated with the ribosomes from infected cells.

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.

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.

Inhibition of host protein synthesis in vaccinia virus-infected cells in the presence of cordycepin (3'-deoxyadenosine).

Cordycepin inhibited efficiently viral mRNA and polyadenylic acid syntheses in vaccinia virus-infected cells, but allowed the shutoff of host protein synthesis to occur. Therefore, cordycepin was used to study this shutoff in the absence of gene expression. Ribosome transit time was increased in infected cells, revealing an inhibition at the level of elongation and/or release of polypeptide chains. However, the disappearance of heavy polysomes in vaccinia virus-infected cells showed that the inhibition of host protein synthesis resulted predominantly from a block at the stage of initiation. This conclusion was confirmed by the recovery of heavy polyribosomes when low levels of cycloheximide were added to slow down ribosome release from the mRNA. Similar amounts of cellular mRNA (present in the polyribosomes) were found in vaccinia virus-infected cells and in mock-infected cels (exposed to cordycepin), showing that the cellular mRNA was not inactivated in these conditions. It was concluded that a component of the vaccinia virion inhibits, in the absence of viral RNA and polyadenylic acid syntheses, host protein synthesis at the level of initiation and, to a lesser extent, at the level of elongation (and/or release) of polypeptide chains.

Expression of vaccinia virus early mRNA in Ehrlich ascites tumor cells. 2. Part of the polysomes at an early stage of virus infection are not bound to the cytoskeleton.

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.