Monoclonal antibodies to two glycoproteins of herpes simplex virus type 2.

Monoclonal antibodies to herpes simplex virus type 2 were found to precipitate different numbers of radiolabeled polypeptides from lysates of virus-infected cells. Antibodies directed against two viral glycoproteins were characterized. Antibodies from hybridoma 17 alpha A2 precipitated a 60,000-molecular-weight polypeptide which chased into a 66,000- and 79,000-molecular-weight polypeptide. All three polypeptides labeled in the presence of [3H]glucosamine and had similar tryptic digest maps. The 60,000-molecular-weight polypeptide also chased into a 31,000-molecular-weight species which did not label with [3H]glucosamine. Antibodies from hybridoma 17 beta C2 precipitated a 50,000-molecular-weight polypeptide which chased into a 56,000- and 80,000-molecular weight polypeptide. These polypeptides also shared a similar tryptic digest map and labeled with [3H]glucosamine. Both monoclonal antibodies were herpes simplex virus type 2 specific. The viral proteins precipitated by 17 alpha A2 antibodies had characteristics similar to those reported for glycoprotein E, whereas the proteins precipitated by 17 beta C2 antibodies appeared to represent a glycoprotein not previously described. This glycoprotein should be tentatively designated glycoprotein F.     

Cellular proteins reactive with monoclonal antibodies directed against simian virus 40 T-antigen.

Several recently isolated monoclonal antibodies which reacted with simian virus 40 T antigens also reacted with proteins found in uninfected and untransformed cells. The proteins were different from each other, PAb419 reacting with a 35,000-molecular-weight protein, PAb427 reacting with a 75,000-molecular-weight phosphoprotein, PAb405 reacting with a 150,000-molecular-weight phosphoprotein, and PAb204 reacting with a 68,000-molecular-weight protein. It is suggested that although some of these cross-reactions may be fortuitous, they may, as an alternative, reflect similarities of shape and perhaps function between domains of the viral T antigen and the relevant host proteins.     

Identification with monoclonal antibodies of virus-specific DNA-binding proteins in the nuclei of cells infected with three serotypes of Marek''s disease virus-related viruses.

Two groups of virus-specific polypeptides were identified in the nuclei of infected cells by cross-reacting monoclonal antibodies with three serotypes of Marek's disease virus. Of these, a 135,000-molecular-weight polypeptide common to all three serotypes was found to bind to both double-stranded and single-stranded DNAs.     

Expression of adenovirus E1B mutant phenotypes is dependent on the host cell and on synthesis of E1A proteins.

Adenovirus mutants containing genetic alterations in the gene encoding the E1B 19,000-molecular-weight (19K) tumor antigen induce the degradation of host cell chromosomal DNA (deg phenotype) and enhanced cytopathic effect (cyt phenotype) after infection of HeLa and KB cells. The deg and cyt phenotypes are a consequence of viral early gene expression in the absence of the E1B 19K protein. The role of the E1A proteins in induction of the cyt and deg phenotypes was investigated by constructing E1A-E1B double mutant viruses. Viruses were constructed to express the individual E1A 13S, 12S, or 9S cDNA genes in the presence of a mutation in the gene encoding the E1B 19K tumor antigen. Expression of either the 13S or 12S E1A proteins in the absence of functional E1B 19K protein produced the deg and cyt phenotypes. In contrast, a virus which expressed exclusively the 9S E1A gene product in the absence of the E1B 19K gene product did not induce the deg and cyt phenotypes, even at high multiplicities of infection. Therefore, both the 13S and 12S E1A gene products could directly or indirectly cause the deg and cyt phenotypes during infection of HeLa cells with an E1B 19K gene mutant virus. Furthermore, the deg phenotype was found to be host cell type specific, occurring in HeLa and KB cells but not in growth-arrested human WI38 cells. These results indicate that expression of the E1A trans-activating and transforming proteins is necessary for the induction of the cyt and deg phenotypes and that host cell factors also play a role.     

Complex of simian virus 40 large-T antigen and host 53,000-molecular-weight protein in monkey cells.

Mouse cells transformed by simian virus 40 (SV40) have been shown to contain a complex of the virus-coded large-T antigen with a host 53,000-molecular-weight (53K) protein. Initial attempts to detect a similar complex in lytically infected cells were unsuccessful, and it therefore seemed that the complex might be peculiar to transformed or abortively transformed nonpermissive cells. Immunoprecipitation of [32P]phosphate-labeled extracts of SV40-infected CV-1 African green monkey kidney cells with antibodies specific for large-T or the 53K protein revealed that the large-T-53K protein complex was formed during lytic infections. Only a minor fraction of the large-T present was associated with 53K protein, and large-T and the 53K host protein cosedimented during centrifugation through sucrose gradients. We used monospecific sera and monoclonal antibodies to study the rate of synthesis and phosphorylation of the 53K protein during lytic infections. Infection of CV-1 cells with SV40 increased the rate of synthesis of the 53K protein fivefold over that in mock-infected cells. At the same time, the rate of phosphorylation of the 53K protein increased more than 30-fold compared with control cultures. Monkey cells transformed by UV-irradiated SV40 (Gluzman et al., J. Virol. 22:256-266, 1977) also contained the large-T-53K protein complex. The formation of the complex is therefore not a peculiarity of SV40-transformed rodent cells but is a common feature of SV40 infections.     

Identification of p34 and p13, human homologs of the cell cycle regulators of fission yeast encoded by cdc2+ and suc1+

cdc2+ and CDC28 play central roles in the cell division cycles of the widely divergent yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. The genes encode protein kinases that show 62% protein sequence identity and are capable of cross-complementation. Monoclonal antibodies were raised against p34cdc2, and a subset recognize p36cdc28. The cross-reacting antibodies detected a 34 kd homolog of the p34cdc2/p36CDC28, protein in HeLa cells. Human p34 was also recognized by an affinity-purified polyclonal anti-p34cdc2 serum. Peptide mapping of p34cdc2, p36CDC28, and human p34 revealed complete conservation of four tryptophan residues in the three proteins. p34 thus appears to be closely related to the two yeast proteins. In addition, a p34 immune complex showed protein kinase activity in vitro, and HeLa cell p34 interacts with p13, the human homolog of the suc1+ gene product of S. pombe.     

Strain variation and nuclear association of Newcastle disease virus matrix protein.

Five monoclonal antibodies to the matrix (M) protein of Newcastle disease virus (NDV) Australia-Victoria (AV) strain were generated and characterized. In competitive antibody-binding assays, the antibodies fell into three discrete groups. The antigenic sites described by these antibody groups were designated M1, M2, and M3. Each antibody reacted with a panel of NDV strains in a manner unique to its group, confirming the grouping by competitive antibody binding. Only site M1 was found on all 12 of the strains tested and may be a "pan-NDV" epitope. A large portion of the M protein of strain AV was detected in the nuclei of infected cells by all five monoclonal antibodies. In addition, the antibodies only stained the nuclei of cells infected with NDV strains expressing M protein containing the corresponding antigenic site. These results confirm that the immunoreactivity in the nucleus is actually caused by the M protein and not by a cross-reacting host protein induced by viral infection.     

IgG autoantibodies against cellular p72 antigen crossing with (MLV) p15-gag antigen: presence in early HIV 1 infection, in HBV infection and in primary Gougerot-Sj?gren]

IgG antibodies of autoimmune SLE (Systemic Lupus Erythematosus) serum S detected a HeLa hnRNP 72 kDa protein, cross-reacting with the retroviral (MLV) p15-gag polypeptide. Since serum S disclosed a ubiquitous 72 kDa antigen in HeLa cell fractions, was prepared the so-called cytoplasmic "X fraction", enriched for the 72 kDa protein, defined here as p72. This autoantigen was detected by antibodies of HIV 1+ patients, recently of seroconverted (RSC) asymptomatic subjects, of HBV+ sera, and of primary Gougerot-Sj?gren (prGS) sera. The presence of these autoantibodies in different autoimmune and infectious pathologies raises the question of the involvement of p72 in the immune processes and in the early HIV1 infection.     

The ebg operon consists of at least two genes

The ebg operon of Escherichia coli includes a second gene designated ebgB. The ebgB gene product is a 79,000-molecular-weight protein and is expressed coordinately with the ebgA gene product, ebg beta-galactosidase. Insertion of the transposable elements Tn5 and Tn9 into ebgA eliminates the expression of ebgB, suggesting that ebgB is distal to ebgA. Ultraviolet light mapping confirms that gene order. The function of the ebgB gene product is unknown.     

Protein product of proto-oncogene c-mil.

Using antipeptide antibodies with specificity for the carboxyl termini of v-raf and v-mil protein products, two proteins with apparent molecular weights of approximately 71,000/73,000 and 215,000 were detected in immunoprecipitates from normal uninfected chicken cells. The 71,000/73,000-molecular-weight protein was identified as the product of the c-mil proto-oncogene by the close structural relationship of its 42,000-molecular-weight carboxyl-terminal domain to the v-mil-encoded domain of the hybrid protein p100gag-mil specified by the avian retrovirus MH2. The amino-terminal domain of the cellular protein is encoded by 5' c-mil sequences that have not been transduced into the genome of MH2. The c-mil protein (p71/73c-mil) was found to be phosphorylated in vivo, and homologous proteins were detected at variable levels in a variety of vertebrate cells, including human cells.     

S gene product: identification and membrane localization of a lysis control protein.

The product of the bacteriophage S gene has been previously shown to be required for an essential step in triggering host cell lysis. By using two different protein labeling systems, maxicells and UV-irradiated infected cells, we identified the S gene product as an 8,500-molecular-weight polypeptide associated with the cell envelope. The apparent molecular weight is significantly less than the 11,500 predicted from the S gene sequence. We were unable to confirm two previous identifications of S gene products, an acidic 15,000-molecular-weight polypeptide found by two-dimensional gel electrophoresis of infected cells and a 5,500-molecular-weight polypeptide in purified phage particles.     

UV light induction of proteins in Bacteroides fragilis under anaerobic conditions

Far-UV irradiation of Bacteroides fragilis cells under anaerobic conditions resulted in the induction of a new 95,000-molecular-weight protein and the increased synthesis of two proteins with molecular weights of 90,000 and 70,000. The latter two proteins were synthesized in small amounts in unirradiated cells. The induction of a 37,000- to 40,000-molecular-weight protein was not observed in irradiated B. fragilis cells. Caffeine, which affected the survival of irradiated B. fragilis cells and reduced host cell-mediated UV reactivation, specifically inhibited the induction of the 95,000-, 90,000-, and 70,000-molecular-weight proteins. Sodium arsenite did not affect the induction of the three inducible proteins or the survival of irradiated B. fragilis cells.     

Cell density-dependent increase in prolyl hydroxylase activity in cultured L-929 cells requires de novo protein synthesis.

Prolyl hydroxylase activity in cultured L-929 cells was found to increase when cells grew from log phase to stationary phase and when cells were harvested at the mid-log phase and replated at higher cell densities. Cycloheximide and actinomycin D inhibited the cell density-dependent increase in prolyl hydroxylase activity indicating that the increase in prolyl hydroxylase activity required de novo synthesis of protein and RNA. Prolyl hydroxylase was purified from cultured L-929 cells and antibodies against the protein were raised in rabbits. The antibodies were used to demonstrate that L-929 cells contained two forms of prolyl hydroxylase: an enzymatically active, tetrameric form consisting of two alpha and two beta polypeptide chains and an enzymatically inactive form containing immunologically cross-reacting protein. The polypeptide chains alpha, beta and cross-reacting protein were obtained by immunoadsorption. Peptide map analysis indicated that cross-reacting protein was similar if not identical to beta in primary structure, and alpha was different from both beta and cross-reacting protein. The results suggested that the prolyl hydroxylase levels in cells or tissues may be regulated by new protein and/or RNA synthesis.     

Mitochondrial binding of a protein affected in mutants resistant to the microtubule inhibitor podophyllotoxin

Specific antibodies to a protein P1 Mr approximately equal to 63,000) from Chinese hamster ovary cells, which is affected in mutants resistant to the microtubule inhibitor, podophyllotoxin, and behaves like a microtubule-related protein by certain criteria [14], have been raised. The antibody reacts specifically with the P1 protein in one- and two-dimensional immunoblots, and a cross-reacting protein of similar molecular mass and electrophoretic mobility is also found in cells from various vertebrate and invertebrate species. The observed similarity in the peptide maps of the cross-reacting protein from human, mouse, Chinese hamster and chicken cells indicates that the structure of this protein should be highly conserved. However, no P1-antibody cross-reacting protein was observed in plants (corn, mung), fungus (Neurospora crassa), yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli and Salmonella typhimurium). Immunofluorescence studies with the P1-antibody show that, in interphase cells of various cross-reacting species, it bound specifically to mitochondria which were associated and distributed on and along the length of microtubules. Similar association and codistribution of mitochondria and microtubules were not observed in mitotic cells. Some implications of the mitochondrial localization of the protein P1 and the observed association between microtubules and mitochondria are discussed.     

Functional complementation of the adenovirus E1B 19-kilodalton protein with Bcl-2 in the inhibition of apoptosis in infected cells.

Expression of the adenovirus E1A oncogene induces apoptosis which impedes both the transformation of primary rodent cells and productive adenovirus infection of human cells. Coexpression of E1A with the E1B 19,000-molecular-weight protein (19K protein) or the Bcl-2 protein, both of which have antiapoptotic activity, is necessary for efficient transformation. Induction of apoptosis by E1A in rodent cells is mediated by the p53 tumor suppressor gene, and both the E1B 19K protein and the Bcl-2 protein can overcome this p53-dependent apoptosis. The functional similarity between Bcl-2 and the E1B 19K protein suggested that they may act by similar mechanisms and that Bcl-2 may complement the requirement for E1B 19K expression during productive infection. Infection of human HeLa cells with E1B 19K loss-of-function mutant adenovirus produces apoptosis characterized by enhanced cytopathic effects (cyt phenotype) and degradation of host cell chromosomal DNA and viral DNA (deg phenotype). Failure to inhibit apoptosis results in premature host cell death, which impairs virus yield. HeLa cells express extremely low levels of p53 because of expression of human papillomavirus E6 protein. Levels of p53 were substantially increased by E1A expression during adenovirus infection. Therefore, E1A may induce apoptosis by overriding the E6-induced degradation of p53 and promoting p53 accumulation. Stable Bcl-2 overexpression in HeLa cells infected with the E1B 19K- mutant adenovirus blocked the induction of the cyt and deg phenotypes. Expression of Bcl-2 in HeLa cells also conferred resistance to apoptosis mediated by tumor necrosis factor alpha and Fas antigen, which is also an established function of the E1B 19K protein. A comparison of the amino acid sequences of Bcl-2 family members and that of the E1B 19K protein indicated that there was limited amino acid sequence homology between the central conserved domains of E1B 19K and Bcl-2. This domain of the E1B 19K protein is important in transformation and regulation of apoptosis, as determined by mutational analysis. The limited sequence homology and functional equivalency provided further evidence that the Bcl-2 and E1B 19K proteins may possess related mechanisms of action and that the E1B 19K protein may be the adenovirus equivalent of the cellular Bcl-2 protein.     

Conformation-dependent antigenic determinants in the toxic lectin ricin.

The major part of the ricin-precipitable antibodies in sera produced by immunizing rabbits with formaldehyde-treated ricin is precipitated also by the isolated ricin A and B chains. In contrast, in antisera produced by immunizing with formaldehyde-treated ricinus agglutinin only a small part of the antibodies cross-reacting with ricin can be precipitated by the isolated A and B chains, or bound to immunoabsorbents containing the isolated ricin chains. In immunodiffusion studies with anti-ricinus agglutinin sera, a star-shaped precipitate was formed when isolated A and B chains recombined to form intact ricin. Both anti-ricin and anti-ricinus agglutinin sera neutralized effectively the ability of ricin to inhibit protein synthesis in HeLa cells. Anti-ricin serum also neutralized the inhibitory effect of the isolated A chain on protein synthesis in a cell-free system and the ability of the isolated B chain to induce indirect hemagglutination. In contrast, antiricinus agglutinin serum did not neutralize the biologic activities of the isolated ricin A and B chains. Anti-ricinus agglutinin serum formed a precipitate with the hybrid ricin A chain/abrin B chain, and protected against the toxic effect on HeLa cells of this hybrid, indicating conformational changes of ricin A chain upon binding to the B chain. It is concluded that the anti-ricinus agglutinin serum contains antibodies directed against conformational determinants present on intact ricin, but not present or exposed in the isolated A and B chains. At least part of these conformational determinants appears to be carried by the A chain.     

Coronavirus JHM: Cell-Free Synthesis of Structural Protein p60

Sac(-) cells infected with murine coronavirus strain JHM shut off host cell protein synthesis and synthesized polypeptides with molecular weights of 150,000, 60,000, and 23,000. The 60,000- and 23,000-molecular-weight polypeptides comigrated with virion structural proteins p60 and p23, and the 60,000-molecular-weight protein was identified as p60 by tryptic peptide fingerprinting. Polyadenylate-containing RNA [poly(A) RNA] extracted from the cytoplasm of infected cells directed the synthesis of both 60,000- and 23,000-molecular-weight polypeptides in messenger-dependent cell-free systems derived from mouse L-cells and rabbit reticulocytes. The reticulocyte system also synthesized a 120,000-molecular-weight polypeptide that was specifically immunoprecipitated by antiserum raised against JHM virions. The identity of the 60,000- and 23,000-molecular-weight in vitro products was established by comigration with virion proteins, immunoprecipitation, and in the case of p60, tryptic peptide fingerprinting. The cytoplasmic poly(A) RNAs which encoded p60 and p23 sedimented in sucroseformamide gradients at 17S and 19S, respectively, and were clearly separable. These RNAs were among the major poly(A) RNA species synthesized in the cytoplasm of actinomycin D-treated cells late in infection, and the in vitro translation of size-fractionated RNA released from polysomes confirmed that they represent physiological mRNA's. These results suggest that the expression of the coronavirus JHM genome involves more than one subgenomic mRNA.     

Chlamydia psittaci IncA is phosphorylated by the host cell and is exposed on the cytoplasmic face of the developing inclusion

Chlamydiae are obligate intracellular bacteria that replicate within a non-acidified vacuole called an inclusion. Chlamydia psittaci (strain GPIC) produces a 39 kDa protein (IncA) that is localized to the inclusion membrane. While IncA is present as a single 39 kDa species in purified reticulate bodies, two additional higher M _r forms are found in C. psittaci -infected cells. This finding suggested that IncA may be post-translationally modified in the host cell. Here we present evidence that IncA is a serine/threonine phosphoprotein that is phosphorylated by host cell enzymes. This conclusion is supported by the following experimental findings: (i) treatment of infected cells with inhibitors of host cell phosphatases or kinases altered the electrophoretic migration pattern of IncA; (ii) treatment with calf intestinal alkaline phosphatase eliminated the multiple-banding pattern of IncA, leaving only the protein band with the lowest relative molecular weight; and (iii) radioimmunoprecipitation of lysates of [³²P]-orthophosphate-labelled infected HeLa cells with anti-IncA antisera demonstrated that the two highest M _r IncA bands were phosphorylated. A vaccinia-virus recombinant expressing incA was used to determine if HeLa cells can phosphorylate IncA in the absence of a chlamydial background. IncA in lysates of these cells migrated identically to that seen in C. psittaci -infected cells, indicating the host cell was responsible for the phosphorylation of the protein. Microinjection of fluorescently labelled anti-IncA antibodies into C. psittaci -infected HeLa cells resulted in immunostaining of the outer face of the inclusion membrane. Collectively, these results demonstrate that IncA is phosphorylated by the host cell, and regions of IncA are exposed at the cytoplasmic face of the inclusion.     

Deletion of the gene encoding the adenovirus 5 early region 1b 21,000-molecular-weight polypeptide leads to degradation of viral and host cell DNA.

The adenovirus 5 mutant H5dl337 lacks 146 base pairs within early region 1B. The deletion removes a portion of the region encoding the E1B 21,000-molecular-weight (21K) polypeptide, but does not disturb the E1B-55K/17K coding region. The virus is slightly defective for growth in cultured HeLa cells, in which its final yield is reduced ca. 10-fold compared with wild-type virus. The mutant displays a striking phenotype in HeLa cells. The onset of cytopathic effect is dramatically accelerated, and both host cell and viral DNAs are extensively degraded late after infection. This defect has been described previously for a variety of adenovirus mutants and has been termed a cytocidal (cyt) phenotype. H5dl337 serves to map this defect to the loss of E1B-21K polypeptide function. In addition to its defect in the productive growth cycle, H5dl337 is unable to transform rat cells at normal efficiency.     

Nuclear organization of splicing small nuclear ribonucleoproteins in adenovirus-infected cells.

We have studied the effect of adenovirus infection on the nuclear organization of splicing small nuclear ribonucleoproteins (snRNPs) in HeLa cells. In uninfected HeLa cells, snRNPs are widespread throughout the nucleoplasm but also are concentrated in specific nuclear structures, including coiled bodies, interchromatin granules, and perichromatin fibrils. We have used immunofluorescence microscopy to study the localization of splicing snRNPs relative to centers of viral DNA synthesis and accumulation identified with antiserum against the viral 72,000-molecular-weight single-stranded DNA-binding protein (72K protein). Splicing snRNPs were independently detected with both monoclonal and polyclonal antibodies specific for common snRNP antigens, snRNP-specific proteins, and the snRNA-specific 2,2,7-trimethylguanosine 5' cap structure. We have examined infected cells 2 to 24 h after infection, and, in the majority of these cells, we observed no colocalization of the snRNP and 72K-protein staining patterns. In the late phase, snRNPs were found to markedly concentrate in discrete clusters that were distinct from the centers of viral DNA synthesis and accumulation identified with anti-72K protein. We have treated cells with hydroxyurea at various times after infection to inhibit aspects of the virus infectious program. We have found that the accumulation of snRNP clusters is correlated with late gene expression rather than with DNA synthesis or early gene expression. Finally, we show that the late-phase snRNP clusters colocalize with a monoclonal antibody that primarily stains interchromatin granules. These results suggest that the centers of snRNP concentration in late-phase infected cells are likely to correspond to interchromatin granule clusters.