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Langerhans cells (LC) are epidermal dendritic cells which express several surface antigens, among them the CD4 antigens. Recent data demonstrated that LC constitute target and storage cells for HIV. To better understand the interactions between HIV and LC, we investigated, in the present work, the fate of HIV envelope glycoproteins (gp120 and gp160) incubated with healthy human trypsinized LC in suspensions. After trypsin treatment, only the epitope for OKT4 appeared to be resistant on LC. In the absence of antigenic sites identified by OKT4A, Leu3a or BL4 (epitopes implicated in HIV binding), LC bound and internalized recombinant HIV gp120 or gp160. This finding supports the hypothesis that there exists at the surface of LC a second molecule which may act as an HIV receptor.

Vaccinia virus, strain WR, was propagated in HeLa cells, L mouse fibroblats, or primary chicken embryo fibroblasts in the presence of [5- (3)H]uridine. Carefully purified virions were found to contain significant amounts of labeled trichloroacetic acid-precipitable material which was rendered acid soluble when digested with pancreatic RNase or hydrolyzed in alkali. Controlled degradation of virions with Nonidet P-40 and 2-mercaptoethanol demonstrated that 65 to 80% of the [5- (3)H]uridine-labeled molecules resided in the viral core. When the total nucleic acids were extracted from viral cores prepared from virions propagated in HeLa cells, 30 to 50% of the total incorporated [5- (3)H]uridine was found in RNA; in L mouse fibroblasts, 40 to 50%; in primary chicken embryo fibroblasts, 50 to 60%. The RNA molecules do not appear to be covalently linked to the viral DNA genome but sediment in sodium dodecyl sulfate-sucrose gradients as 8 to 10S species relative to ribosomal RNA.

Restriction endonucleases EcoRI, BamHI, Hind III and KpnI were used for analysis of acccinia virus DNA. The number and size of restriction endonuclease fragments were determined by gel electrophoresis and the analysis of 3H-labeled vaccinia virus DNA. It was shown that many EcoRI and BamHI fragments had the same and similar sizes. The exact number of EcoRI and BamHI fragments were estimated only after analysis of [3H]-labeled vaccinia DNA. The vaccinia genome sizes calculated from HindIII, KpnI and EcoRI are very close to the actual genome weight. But the sum of BamHI fragments is much lower than those determined by electron microscope method.

Vaccinia virus DNA fragments that have been denatured by alkali and then neutralized contain a fraction that rapidly reforms duplex structures. The fraction is enriched by fractionating on hydroxyapatite columns and serves as as substrate for digestion by two restriction endonucleases isolated from Hemophilus parainfluenzae, Hpa I and HPa II. The patterns obtained by gel electrophoresis of the digested fragments show the presence of three major bands after Hpa I digestion and four major bands after Hpa II digestion. The DNA that is isolated from some of these bands quickly reforms duplex regions after alkaline denaturation. The size of the DNA segments in the major bands has been estimated to be in the range of 0.44 X 10(6) to 3.2 X 10(6) daltons. The fragments which rapidly reform duplex chains after denaturation are sensitive to single-strand-specific nucleases. These results are consistent with a model of vaccinia virus DNA which has a covalent link connecting complementary chains.

In both rabbit poxvirus and vaccinia virus DNA have demonstrated an identical distribution of eight HinfI. The length of the terminal repeats was found to be 3.4 to 3.6 megadaltons (Mdaltons) for rabbit poxvirus DNA and 7.4 to 8.0 Mdaltons for vaccinia virus DNA. Maps of the HinfI restriction sites within isolated EcoRI end fragments of rabbit poxvirus and vaccinia virus DNA PHAVE DEMONSTRATED AN IDENTICAL DISTRIBUTION OF EIGHT HinfI sites in an internal part (approximately 2 Mdaltons) of the EcoRI end fragments of the two genomes.

Structural polypeptides from a number of poxviruses were analysed by SDS-polyacrylamide gel electrophoresis. The patterns obtained with orthopoxviruses were generally quite similar to one another, but variola, monkeypox, cowpox and vaccinia viruses could be distinquished by their profiles in the molecular weight (mol. wt.) region around 30,000 to 40,000; some additional variation was found amongst cowpox and vaccinia strains. Whitepox virus was shown to have structural polypeptides indistinguishable from those of variola virus. The structural polypeptides of poxviruses belonging to other genera were different from those of the orthopoxvirus, except those of mol. wt. about 122,000 and 97,000, which were common to all viruses irrespective of genus. A polypeptide of mol. wt. about 25,000 was also observed in all cases, though its position varied slightly with the individual virus.

A major vaccinia virus core protein, designated VP8, has been purified from virions to homogeneity through DEAE-cellulose, CM-cellulose, and hydroxyapatite chromatography. VP8 migrates as a 25-kDa band in SDS-polyacrylamide gel electrophoresis and sediments as a monomeric species in neutral sucrose gradient centrifugation. This protein is a significant constitutent of the virion, comprising about 6.5% of the total viral polypeptides by mass. Analysis by filter binding and by sucrose gradient centrifugation shows that VP8 binds to double-stranded as well as to single-stranded DNA at low salt concentrations (25 mM NaCl). At higher salt concentrations (100 mM NaCl), the protein binds with a relatively greater affinity to single-stranded DNA. The results from sucrose gradient centrifugation indicate that VP8 probably binds noncooperatively to all structural forms of DNA. The protein is likely to be a component of the viral nucleoprotein complex.

The protein synthesis inhibitor previously demonstrated to be associated with vaccinia cores was quantitatively solubilized from vaccinia virions or cores after an endogenous protein kinase reaction at pH 10. The presence of the inhibitor in the soluble extract correlated with the presence of soluble virion proteins phosphorylated in vitro. Partially purified inhibitor blocked methionyl-tRNAfMet-40S initiation complex formation, as was the case in cell-free extracts derived from vaccinia virus-infected cells.

The sequence arrangement within the nontranscribed portion of the inverted terminal repetition of the vaccinia virus genome exists in quasi-stable and unstable forms that are not distinguishable on the basis of viral infectivity. The unstable forms, which composed about 20% of a serially passaged stock of virus, were recognized by terminal heterogeneity on restriction endonuclease analysis. Instead of a single terminal fragment from each end of the genome, an array of eight or more fragments differing in size by 1650-base-pair increments was detected. This feature was not eliminated by repeated plaque purification, indicating that the population of DNA molecules with various numbers of reiterations can rapidly evolve from the DNA of a single virus particle. However, at each successive round of plaque purification, about 20% of the unstable isolates revert back to the more stable form. Stable forms are characterized by the presence of a set of 13-17 tandem 70-base-pair repeats on each side of a 435-base-pair intervening sequence near both ends of the genome. In contrast, the unstable forms possess sets of tandem repeats and intervening sequences that alternate many times in series. The transition between the two genomic forms and the evolution of the unstable form appear to be mediated by recombinational events.

A restriction endonuclease from Haemophilus influenzae (Hind III) specifically cleaved vaccinia DNA into 14 fragments. The molecular weights of these fragments were determined by gel electrophoresis and ranged from 0.5 x 10(6) to 30 x 10(6). Hind III digestion of the DNA from the WR and CV-1 strains of vaccinia revealed a small molecular difference in one of the resulting fragments. The average molecular weight of the entire vaccinia genome was calculated to be 125 x 10(6).

The poxvirus Cotia was studied by electron microscopy and by serological and biochemical analyses. Thin-sectioned preparations of infected Vero cells indicated that Cotia virus morphogenesis was similar to other mammalian poxviruses; unique filamentous structures and inclusion matrices were apparent in the cytoplasm. Complement fixation tests that included purified Cotia virions showed a reciprocal cross-reaction with rabbit myxoma virus and no cross-reaction with vaccinia virus. Serological results coupled with gradient polyacrylamide gel electropherograms of the structural proteins of purified Cotia, vaccinia, myxoma and fibroma viruses suggested that Cotia virus was similar to the latter two viruses. Agarose gel electropherograms of cleavage fragments of each of these virus DNAs digested with three separate restriction endonucleases showed that each of these viruses had a unique DNA gel profile.

The localization of KpnI, SacI, XhoI, AvaI, PstI, BglI, BamHI, EcoRI, PmiI, SalI, BglII, restriction endonuclease cleavage sites in HindIII-F-fragments of DNA from vaccinia strains WR, Copenhagen, LIVP and neurovaccine has been detected. The fragments have been shown to differ in the number of AvaI, EcoRI and BamHI sites. The fragments also differ from the analogue of Tian Tan vaccinia strain in the pattern of restriction by AvaI, XhoI, PstI, EcoRI and BamHI endonucleases.

Analysis of vaccinia DNA by reassociation kinetics revealed that 7% of the genome contains a sequence repeated 10 times. This sequence does not contain any host cell DNA, is viral specific, and is found in virions passed at either high or low multiplicities of infection.

Cleavage sites for the restriction endonucleases EcoRI, KpnI and XhoI were mapped on rabbit poxvirus and vaccinia virus DNA. These physical maps were used to analyse the structural variations between the two DNAs. Two specific heterologous regions, symmetrically arranged at each end of the genomes, have been identified. Region 1, representing the exterior part of the terminal repetition, appears to contain unrelated sequences in each DNA and accounts for the difference in length of the two genomes. Region 2, separated from region 1 by a conserved part of the terminal repetition, is located at the transition from repeated to unique DNA sequences. Its overall length of about 4 megadaltons is well conserved and it contains individual DNA-specific as well as conserved restriction sites. The major central part of the genomes (over 100 megadaltons) contains very few, widely dispersed restriction site variations.

Data are presented which indicate that vaccinia DNA does not contain poly(dT) sequences the size of poly(A) sequences (50 to 200 nucleotides in length) found in vaccinia RNA. A hybridization experiment and polyacrylamide gel electrophoresis and DEAE-Sephadex chromatography of pyrimidine tracts show that poly(dT) sequences can account for no more than 0.1% of vaccinia DNA. Ultraviolet irradiation (which causes thymine dimer formation) and phleomycin (which binds to thymidine) both inhibit RNA synthesis but not poly(A) synthesis by vaccinia cores. These data are consistent with a nontranscriptive mechanism for vaccinia poly(A) synthesis. Both trypsin and 50 C heat treatment inhibit RNA synthesis more than poly(A) synthesis by cores, suggesting that separate enzymes may be involved in these syntheses. When the rate of core RNA synthesis is reduced by lowering the UTP and GTP concentrations, the size of the poly(A) sequences increase. These and other data suggest that transcription is involved in the termination of poly(A) synthesis in cores. This might be due to the displacement of growing poly(A) chains by recently completed RNA 3' termini which have not yet acquired poly(A) sequences.

In the presence of ATP plus two other ribonucleoside triphosphates or in reactions containing all four ribonucleoside triphosphates and actinomycin D, vaccinia virus synthesizes in vitro discrete low-molecular-weight RNA molecules ranging in size from about 20 to several hundred bases. A novel feature of these small RNA molecules is that they are capped and methylated at the 5' terminus, containing both mGpppGm and mGpppAm type cap structures, and in addition these molecules are polyadenylated at the 3' terminus. Hybridization of these RNAs to restriction fragments derived from vaccinia virus DNA indicates a considerable degree of complexity, suggesting the presence of a large number of promoters throughout the genome. However, measurable sensitivity to pancreatic RNase of the 5' capped end of these RNAs while in hybrid form to the DNA suggests other possible roles for these small RNAs in vaccinia virus mRNA biogenesis.

The coding properties of RNA transcribed in vitro by purified vaccinia cores have been investigated using Krebs ascites tumor cells, L cells, and reticulocyte lysates. Six to 10 proteins synthesized in vitro are separated on polyacrylamide gels by electrophoresis in the presence of sodium dodecyl sulfate. Their molecular weights vary from 10,000 to 44,000. The electrophoretic behavior of these proteins is similar to that of early proteins isolated from infected L cells. The tryptic peptide analysis of one of these proteins indicates similarity in amino acid sequences. These results show fidelity of both in vitro transcription and molecular weight above 44,000 are synthesized in vitro does not seem due to a competition between 12S mRNA synthesized in excess and RNA of a higher sedimentation coefficient present in a lower amount.

Approximately 15% of the polyadenylic acid-containing cytoplasmic RNA labeled from 5 to 7 h after vaccinia virus infection formed intermolecular duplex structures characterized as double-stranded RNA by RNase resistance, density in Cs2SO4, base composition, chromatography on cellulose, and ability to inhibit reticulocyte cell-free protein synthesis. Both sucrose gradient sedimentation and electron microscopic analysis indicated that the double-stranded regions were several hundred to more than a thousand nucleotide base pairs long. The double-stranded RNA, after denaturation, hybridized to approximately 25% of the vaccinia virus genome, whereas total late RNA hybridized to 42%. The finding that the duplex RNA, after denaturation, hybridized to most HindIII restriction endonuclease fragments of vaccinia virus DNA indicated that symmetrical transcription is not confined to the terminal inverted repeat sequence or to one contiguous region of the genome. Although relatively little labeled, early, polyadenylic acid-containing RNA formed RNase-resistant hybrids upon self-annealing, the percentage increased upon addition of unlabeled late RNA, indicating that the latter contains "anti-early" sequences.

As measured by in vitro, 51Cr-release assays, the expression on plasma membranes of two, immediate-early, vaccinia virus-specified cell-surface antigens, with mol wt of 25K-27K and 16K-17K, could be directly correlated with the susceptibility of target cells to lysis by vaccinia virus-specific cytotoxic T cells.

Infectious vaccinia virus can be purified from whole cells by experimentally induced lysis (intracellular virus) or from supernatant growth medium (extracellular virus). Extracellular virus and intracellular virus differed by buoyant density (1.237 versus 1.272 g/cm3), phospholipid content and composition, and polypeptide pattern. Differences in structural polypeptides on the virus surface could be detected by lactoperoxidase-catalyzed radioiodination or Brij treatment. Characteristic of extracellular virus was an additional polypeptide, with a molecular weight of 37,000 (37K), which represented 5 to 7% of the total particle protein. Antibodies to the 37K protein detected only some of the cell-associated particles late in normal infection. Upon treatment of infected cultures with N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine, a drug which prevents vaccinia virus release, no particle-associated 37K protein could be detected. In all other properties tested so far, except for a slight difference in phospholipid composition, the virus obtained in the presence of the drug resembled the normal intracellular virus. N1-Isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine prevented vesicularization of intracellular viral particles. Lack of vesicularization was accompanied by the absence of particle-associated 37K viral protein and seemed to correlate with an inhibition of virus dissemination to the cell periphery.

Orthopoxvirus DNA from representative strains of rabbitpox, vaccinia, monkeypox, variola, cowpox and ectromelia viruses was analysed by cleavage with restriction endonucleases HindIII, XhoI or SmaI. Genome mol. wt. vary from about 120 x 10(6) for rabbitpox to about 145 x 10(6) for cowpox. Physical maps of cleavage sites are similar and characteristic for strains of the same Orthopoxvirus type. The distribution of HindIII sites suggests that an internal region of mol. wt. about 30 x 10(6) is highly conserved between Orthopoxvirus genomes although some type-specific differences occur within this region, especially with strains of ectromelia virus. Conservation of internal sequences is less marked following analysis with XhoI although cleavages within this central region of particular genomes appear to represent a subset of preferred sites. Endonuclease SmaI cleaves exceptionally infrequently and distinguishes variola, monkeypox, vaccinia, cowpox or ectromelia viruses. Type specific differences result largely from extensive, near terminal variations in length and sequence. Representative Orthopoxvirus genomes have rapidly renaturing terminal restriction fragments confirming the presence of near terminal, covalent cross-links. Terminal restriction fragments from the same or different genomes generally cross hybridize indicating the presence of near terminal repetitions of mol. wt. up to 6 x 10(6) and which share at least a subset of common sequences. Variola strains however, appear to lack such sequences from one specific terminus which maps shorter than that of related viruses.

HeLa, SIRC, and RK-13 cells were compared as to their production of intracellular naked vaccinia virus (INV) and extracellular enveloped vaccinia virus (EEV) after infection with vaccinia strains WR and IHD-J. IHD-J produced more EEV from all three cell lines than did WR, although both strains produced approximately the same quantity of INV. The most efficient EEV release was from RK-13 cells infected with IHD-J, which was 200 times more than from WR-infected SIRC cells. This permitted for the first time the purification of milligram quantities of EEV that contained much fewer cell protein contaminants than could be obtained from HeLa or SIRC cells. The INV surface proteins 200K, 95K, 65K, and 13K were present in both HeLa and RK-13 cell-derived INV but were absent in SIRC cell INV. These proteins were absent in EEV from all three cell lines. Four glycoproteins of molecular weights 210 x 10(3) (210K), 110K, 89K, and 42K and five glycoproteins in the 23K to 20K range plus a nonglycosylated protein of 37K were detected in EEV from the hemagglutinin-positive IHD-J vaccinia strain. The 89K glycoprotein was not present in EEV or membranes from cells infected with the hemagglutinin-negative vaccinia strain IHD-W. Antisera to IHD-W lacking hemagglutinin-inhibiting antibodies did not precipitate the 89K glycoprotein of IHD-J. The only glycoprotein that specifically attached to rooster erythrocytes was the 89K glycoprotein. This evidence indicates that the 89K glycoprotein is the vaccinia hemagglutinin.

Two different twodimensional cellulose thinlayer separations for blocked, methylated mRNA 5'-termini are described. They allow rapid analysis even of complex mixtures of mRNA "cap" structures on the basis of their methyl group content and base composition. These simple procedures are especially useful for the analysis of [3H-methyl]-labeled mRNA in combination with tritium fluorography. A qualitative and quantitative analysis of the methylated "cap" structures of in vitro labeled Vaccinia "core" mRNA is presented. The presence of methylated "cap" structures in Vaccinia RNA increases the in vitro translation efficiency of methylated Vaccinia RNA over Vaccinia RNA transcribed in the absence of a methyl group donor.

The DNA of red cowpox virus strain Brighton or its white pock variant was analysed by cleavage with restriction endonucleases HindIII, XhoI, PstI or KpnI. Physical maps were constructed and the genomes compared with that of vaccinia virus strain DIE. The size of the red cowpox genome is 23 to 29 megadaltons greater than that of vaccinia and results from the presence of additional, near terminal sequences. An internal region of about 75 megadaltons appears to be highly conserved between the two viruses. The red cowpox genome contains near terminal, repetitive sequences which have some homology with those of vaccinia virus DNA. Rapid renaturation of red cowpox terminal restriction fragments indicates that these are covalently cross-linked. Viable white pock variants arise continually and map as deletion mutants lacking similar sequences from one specific terminus only of the parental genome. The deletion represents 11 to 12% of the red cowpox DNA and includes the terminal repetition which therefore is not required for replication. The deleted terminus of the white pock variant genome does not appear to be cross-linked.

Vaccinia virions propagated in the presence of [3H]ornithine were found to contain two labeled polyamines, spermine and spermidine. In complete virions the ratio of radioactively labeled spermine to spermidine was about 1:10, whereas in viral cores the ratio was 2:5. This suggests that some spermidine was preferentially lost during the conversion of virions to cores or that spermidine was present in the virions both inside and outside the core structure. Addition of [3H]ornithine to vaccinia virus-infected cells as late as 6 h postinfection demonstrated that, although the conversion of this precursor to polyamines was reduced by 50% or more as compared to mock-infected cells, complete inhibition of polyamine synthesis did not occur. Two percent or less of the total radioactivity associated with virions grown in the presence of [3H]ornithine was found to be acid soluble. Polyacrylamide gel electrophoretic analysis showed that all the structural polypeptides were labeled when virions were propagated in the presence of [3H]ornithine. When cores labeled with a mixture of 14C-labeled amino acids were extracted with 0.25 N H2SO4, 12 to 15% of the labeled core polypeptides were released and could be precipitated with acetone. About 40% of [3H]arginine-labeled polypeptides associated with cores were extracted with acid. Four polypeptides or groups of polypeptides were resolved after polyacrylamide gel electrophoresis analysis of the acid-soluble fraction of cores with molecular weights of about 58,000, 34,000, 24,000 and 10,000 to 12,000. About 40% of the [3H]arginine radioactivity extracted from cores coelectrophoresed with the 10,000 to 12,000-molecular weight polypeptide, indicating that this may represent an arginine-rich, histone-like structural polypeptide of the virion.

Radioactively labeled vaccinia, cowpox and Shope fibroma virions free from any detectable contamination with host cell protein, were dissociated into their constituent polypeptides, and these were then analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The profiles of constituent polypeptide bands of four strains of vaccinia virus (IHD-W, IHD-J, Lister and DIs) were almost the same, except that a polypeptide of about 41,000 daltons was not detectable in the autoradiogram of strain IHD-W which has no hemagglutinin. The profile of polypeptide bands of cowpox virions was also almost the same as that of vaccinia virions, except for several polypeptides of about 40,000 to 50,000 daltons, but the profile of Shope fibroma virions differed considerably from that of vaccinia or cowpox virions.

Vaccinia virus propagated in rotated cultures of RK13 cells was purified by sucrose density gradient zonal centrifugation. The purified virus was dissociated with sodium dodecyl sulfate end its protein composition analyzed by polyacrylamide gel electrophoresis. The protein composition of the virion envelope, isolated by the non-ionic detergent Nonidet P-40, was also determined.

A glycoprotein component of vaccinia virus was extracted with a non-ionic detergent NP-40 (Nonidet P-40) and purified by gel chromatography. The single antigen extracted by the detergent had a molecular weight estimated between 100,000 and 200,000 daltons. This glycoprotein was found to contain less than 1% hexosamine which would correspond to 5--10 sugar residues per molecule. Antibodies produced against this glycoprotein were able to neutralize vaccinia virus. Using immunoelectron microscopy, this molecule was found to be located in the outer layer of the virion. These results further suggest that this protein called complex E (for external) is a surface component of vaccinia virus.

Monoclonal antibodies directed against major antigens induced by cowpox virus (CPV) were produced. The specificities of these antibodies were established by immunoprecipitation, immunoblotting and several serological analyses, and from the cross-reactivities of these antibodies with cells infected with various other poxviruses, ectromelia virus (EV), vaccinia virus and Shope fibroma virus. The antibodies defined included ones reacting with each of the known major antigens of poxviruses, i.e. the common antigen of all poxviruses (probably NP antigen), the Orthopoxvirus-specific antigen (probably LS antigen), the haemagglutinin, the cell surface antigen, the common A-type inclusions in CPV and EV, and the antigen involved in neutralization.