Retinoids and cancer: antitumoral effects of ATRA, 9-cis RA and the new retinoid IIF on the HL-60 leukemic cell line.

OBJECTIVE: To compare the antitumoral effects of all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA) with those of 5-OH,11-O-hydrophenanthrene (IIF), a new derivative of retinoic acid. MATERIALS AND METHODS: The effect of retinoids was tested on cell line HL-60. Cell differentiation and apoptosis were evaluated by morphological and biochemical analysis as BCL-2 protein and by DNA fragmentation assay. The ability to activate retinoic acid receptors (RAR) and/or retinoid X receptors (RXR) and to modulate gene expression was determined by transactivation assay. RESULTS: With cell line HL-60, the antiproliferative effect of IIF was stronger than that of ATRA and 9-cis RA. Following retinoid treatment, cells appeared to differentiate and apoptotic cells were observed. The appearance of DNA laddering and a decrease in the amount of BCL-2 protein confirmed apoptosis. IIF transcriptionally activated RXR-gamma more than RAR-alpha. CONCLUSION: The findings indicate that IIF transcriptionally activates RXR-gamma preferentially, induces apoptosis and has a more antiproliferative activity than ATRA and 9-cis RA on cell line HL-60.

Novel, soluble isoform of the herpes simplex virus (HSV) receptor nectin1 (or PRR1-HIgR-HveC) modulates positively and negatively susceptibility to HSV infection.

A novel member of the nectin family, nectin1gamma, was molecularly cloned. The cDNA has the same ectodomain as nectin1alpha and nectin1beta, the two known transmembrane isoforms that serve as receptors for herpes simplex virus (HSV) entry into human cell lines (nectin1alpha and nectin1beta, also called PRR1-HveC and HIgR, respectively). The 1.4-kb transcript, which originated by alternative splicing, is expressed in human cell lines, and appears to have a narrow distribution in human tissues. The sequence does not have a hydrophobic anchoring region, and the protein is secreted in the culture medium of cells transfected with the cDNA. Nectin1gamma, purified from culture medium, can compete with membrane-bound nectin1beta and reduce HSV infectivity. The expression of nectin1gamma cDNA in cells resistant to HSV infection and lacking HSV receptors enables HSV to enter the cell, which implies that it is present at the cell surface. Thus, nectin1gamma has the potential both to mediate and to reduce HSV entry into cells.

Comparison of murine and human nectin1 binding to herpes simplex virus glycoprotein D (gD) reveals a weak interaction of murine nectin1 to gD and a gD-dependent pathway of entry.

The murine nectin1alpha (mNectin1alpha), a homolog of human nectin1alpha (hNectin1alpha, or PRR1, HveC), mediates the entry of herpes simplex virus (HSV) into cells. Previously, we reported that the binding of hNectin1 to HSV glycoprotein D (gD) was readily detectable, whereas the binding of mNectin1 to gD was not detectable, thus raising the question whether mNectin1 mediates a gD-dependent or a gD-independent pathway of entry. Here we report comparative binding studies of murine- and human-nectin1alpha to virions and to gD. The assays consistently showed either a very weak binding or no detectable binding of murine nectin1alpha to gD. They included (i) binding of soluble mNectin1-Fc or hNectin1-Fc to virions and competition of the binding by soluble gD(Delta290-299t) and by monoclonal antibodies to gD; (ii) pull-down experiments of wt gD from lysates of infected cells; and (iii) ELISA binding of soluble gD(Delta290-299t) to cells expressing mNectin1 or hNectin1. In contrast to the binding studies, the entry studies readily showed that entry mediated by mNectin1 was dependent on gD. Thus, a gDnull (gD-/-) mutant virus was unable to enter mNectin1-expressing cells, and entry of wild-type virus was inhibited by antibodies to gD or soluble gD at similar concentrations. We infer that gD represents a weak ligand in the interaction between mNectin1 and virions, whereas it represents a strong and the major ligand for hNectin1. Yet gD is required in HSV-1 entry mediated by mNectin1alpha. We conclude that a high-affinity binding of the receptor to gD is not a requirement in the gD-dependent pathway of HSV entry to cells.

The murine homolog of human Nectin1delta serves as a species nonspecific mediator for entry of human and animal alpha herpesviruses in a pathway independent of a detectable binding to gD.

The full-length cDNA of the murine homolog of human nectin1delta (mNectin1delta), also known as human poliovirus receptor related 1 (PRR1) or herpesvirus entry mediator C, was cloned and showed a >90% identity with its human counterpart. mNectin1delta is expressed in some murine cell lines, exemplified by NIH 3T3 and L cells, and in murine tissues. It mediates entry of an extended range of herpes simplex virus (HSV) strains, porcine pseudorabies virus (PrV), and bovine herpesvirus 1. A soluble form of the mediator blocked infectivity in mNectin1delta and human nectin1delta (hNectin1delta)-expressing cells, suggesting a physical interaction of the mediator with virions. The higher concentrations of soluble mNectin1 required to block infectivity relative to soluble hNectin1 suggest that the target of the two molecules is not identical. Entry of HSV, but not PrV, was blocked by soluble mNectin1delta in NIH 3T3 and L cells. Two features were unexpected. First, soluble mNectin1delta failed to physically interact with HSV glycoprotein D (gD) at a detectable level, although it interacted physically with virions. Second, coexpression of mNectin1delta and HSV gD did not restrict HSV or PrV infection, whereas coexpression of hNectin and gD did restrict infection, suggesting that mNectin1delta fails to be sequestered by HSV gD. We conclude that mNectin1delta serves as a species-nonspecific mediator for entry of the human and animal alphaherpesviruses. This activity, at least for HSV, is independent of a detectable binding to gD.

Nectin2alpha (PRR2alpha or HveB) and nectin2delta are low-efficiency mediators for entry of herpes simplex virus mutants carrying the Leu25Pro substitution in glycoprotein D.

The receptors for entry of herpes simplex viruses 1 and 2 (HSV-1 and -2), widely expressed in human cell lines, are members of a subset of the immunoglobulin superfamily exemplified by herpesvirus entry mediator C (HveC) and the herpesvirus immunoglobulin-like receptor (HIgR). This report focuses on two members of this subset, herpesvirus entry mediator B (HveB), recently designated nectin2/PRR2alpha, and its splice variant isoform, nectin2/PRR2delta. Nectin2alpha and -delta share the ectodomain but differ in the transmembrane and cytoplasmic regions. HveB was reported to enable entry of HSV-1 carrying mutations in glycoprotein D (gD) and of HSV-2, but not of wild-type (wt) HSV-1. We report that (i) both nectin2alpha and -delta served as receptors for the entry of HSV-1 mutant viruses HSV-1(U10) and -(U21) and AP7(r) that carry the Leu25Pro substitution in gD but not for HSV-1 mutants U30 and R5000 that carry the Ser140 or Ala185 substitution in gD. All of these mutants were able to overcome the block to entry mediated by expression of wt gD. (ii) Infection of cells expressing nectin2alpha or -delta required exposure to multiplicities of infection about 100-fold higher than those required to infect cells expressing HveC or HIgR. (iii) gD from HSV-1(U21) bound in vitro soluble forms of nectin2. The association was weaker than that to the soluble form of HveC/HIgR. Binding of wt HSV-1 gD to soluble nectin2 was not detectable. (iv) A major region of nectin2 functional in virus entry mapped to the V domain, located at the N terminus.

Conservation of the architecture of the Golgi apparatus related to a differential organization of microtubules in polykaryocytes induced by syn- mutants of herpes simplex virus 1.

Infection of Vero and HEp-2 but not of 143TK- cells with herpes simplex virus 1 results in fragmentation and dispersal of the Golgi apparatus. Concurrently, in all three infected cell lines the microtubular network is disrupted, suggesting that the disruption of microtubules is essential but not sufficient to induce the fragmentation of the Golgi apparatus. We now report the following: (i) In polykaryocytes formed in Vero cells infected with HSV-1 syn- mutant viruses, intact Golgi stacks were readily detected by electron microscopy. These aggregated in the center of large polykaryocytes. (ii) The distribution of viral glycoprotein D, examined in both fixed and nonfixed cells, appeared to match the distribution of the Golgi stacks, suggesting that the aggregated Golgi stacks funnel viral glycoproteins and viral particles to a limited region of the plasma membrane of the polykaryocytes rather than directing exocytic flow in a more dispersed fashion as seen in syn+ virus-infected cells exhibiting fragmented and dispersed Golgi. (iii) In most polykaryocytes, the microtubules formed parallel bundles extending along the axis of recruitment of new cells. (iv) Fragmentation of the microtubules at the periphery of the cell near the plasma membrane was observed in untreated or cycloheximide-treated cells 2 h after infection with syn- virus HSV-1(MP) or syn+ HSV-1(mP) but not in mock-infected cells. These observations suggest that peripheral depolymerization is initiated at the time of infection and that a factor which determines the syn- or syn+ phenotype is whether the microtubular network regenerates concomitant with cell fusion or reorganizes to form a collapsed network surrounding nuclei of syn+ infected cells.

CD68+ cells of monocyte/macrophage lineage in the environment of AIDS-associated and classic-sporadic Kaposi sarcoma are singly or doubly infected with human herpesviruses 7 and 6B.

Earlier studies have shown that Kaposi sarcomas contain cells infected with human herpesvirus (HHV) 6B, and in current studies we report that both AIDS-associated and classic-sporadic Kaposi sarcoma contain HHV-7 genome sequences detectable by PCR. To determine the distribution of HHV-7-infected cells relative to those infected with HHV-6, sections from paraffin-embedded tissues were allowed to react with antibodies to HHV-7 virion tegument phosphoprotein pp85 and to HHV-6B protein p101. The antibodies are specific for HHV-7 and HHV-6B, respectively, and they retained reactivity for antigens contained in formalin-fixed, paraffin-embedded tissue samples. We report that (i) HHV-7 pp85 was present in 9 of 32 AIDS-associated Kaposi sarcomas, and in 1 of 7 classical-sporadic HIV-negative Kaposi sarcomas; (ii) HHV-7 pp85 was detected primarily in cells bearing the CD68 marker characteristic of the monocyte/macrophage lineage present in or surrounding the Kaposi sarcoma lesions; and (iii) in a number of Kaposi sarcoma specimens, tumor-associated CD68+ monocytes/macrophages expressed simultaneously antigens from both HHV-7 and HHV-6B, and therefore appeared to be doubly infected with the two viruses. CD68+ monocytes/macrophages infected with HHV-7 were readily detectable in Kaposi sarcoma, but virtually absent from other normal or pathological tissues that harbor macrophages. Because all of the available data indicate that HHV-7 infects CD4+ T lymphocytes, these results suggest that the environment of the Kaposi sarcoma (i) attracts circulating peripheral lymphocytes and monocytes, triggers the replication of latent viruses, and thereby increases the local concentration of viruses, (ii) renders CD68+ monocytes/macrophages susceptible to infection with HHV-7, and (iii) the combination of both events enables double infections of cells with both HHV-6B and HHV-7.

Identification of an 85 kDa phosphoprotein as an immunodominant protein specific for human herpesvirus 7-infected cells.

The reactivity of human cord blood sera was directed most frequently in Western blot assays to a protein with an apparent molecular mass of 85 kDa that belongs to the p85 complex, a family of antigenically related proteins identified previously in our laboratory with the aid of two MAbs. We show that the 85 kDa protein is phosphorylated. As antibodies present in the human sera were directed in part to proteins carrying cross-reactive epitopes between human herpesvirus 6 (HHV-6) and 7 (HHV-7), it is remarkable that reactivity to the 85 kDa phosphoprotein was maintained after preabsorption of the sera with HHV-6 antigen, but abolished after preabsorption with HHV-7 antigen. Therefore, the 85 kDa phosphoprotein may be considered a major determinant of the human immune response to HHV-7, discriminating HHV-6 from HHV-7 infection.

Redistribution of microtubules and Golgi apparatus in herpes simplex virus-infected cells and their role in viral exocytosis.

Earlier studies have shown that the Golgi apparatus was fragmented and dispersed in herpes simplex virus 1-infected Vero and HEp-2 cells but not in human 143TK- cells, that the fragmentation and dispersal required viral functions expressed concurrently with or after the onset of DNA synthesis (G. Campadelli-Fiume, R. Brandimarti, C. Di Lazzaro, P. L. Ward, B. Roizman, and M. R. Torrisi, Proc. Natl. Acad. Sci. USA 90:2798-2802, 1993), and that in 143TK- cells, but not Vero or HEp-2 cells, infected with viral mutants lacking the UL20 gene virions were glycosylated and transported to extracellular space (J. D. Baines, P. L. Ward, G. Campadelli-Fiume, and B. Roizman, J. Virol. 65:6414-6424, 1991; E. Avitabile, P. L. Ward, C. Di Lazzaro, M. R. Torrisi, B. Roizman, and G. Campadelli-Fiume, J. Virol. 68:7397-7405, 1994). Experiments designed to elucidate the role of the microtubules and of intact or fragmented Golgi apparatus in the exocytosis of virions showed the following. (i) In all cell lines tested (Vero, 143TK-, BHK, and Hep-2) microtubules underwent fragmentation particularly evident at the cell periphery and then reorganized into bundles which circumvent the nucleus. This event was not affected by inhibitors of viral DNA synthesis. We conclude that redistribution of microtubules may be required but is not sufficient for the fragmentation and dispersal of the Golgi apparatus. (ii) In all infected cell lines tested, nocodazole caused fragmentation and dispersal of the Golgi and a far more extensive depolymerization of the microtubules than was seen in untreated, infected Vero or HEp-2 cells. Taxol precluded the depolymerization of the microtubules and fragmentation of the Golgi in both infected cell lines. Neither nocodazole nor taxol affected the exocytosis of infectious virus from Vero, HEp-2, or 143TK- cells infected with wild-type virus. We conclude that the effects of nocodazole or of taxol are dominant over the effects of viral infection in the cell lines tested and that viral exocytosis is independent of the organization of microtubules or of the integrity of the Golgi apparatus. Lastly, the data suggest that herpes simplex viruses have evolved an exocytic pathway for which the UL20 protein is a component required in some cells but not others and in which this protein does not merely compensate for the fragmentation and dispersal of the Golgi apparatus.

Selection of a monoclonal antibody specific for variant B human herpesvirus 6-infected mononuclear cells.

A monoclonal antibody, designated as MAb 6E2, specific for human herpesvirus 6 variant B (HHV-6B) was derived from the spleen of a mouse immunized with lysates of HHV-6B(Z29) cord blood mononuclear cells. MAb 6E2 reacts by immunofluorescence with all the HIV-6B strains tested (Z29, CV, Hashimoto and SF) and fails to react with variant A prototypes, GS and U1102. The immunofluorescence staining was punctate and localized to the cytoplasm. The protein reacting with MAb 6E2 was identified as protein 48,000 in apparent M(r) value by immunoaffinity chromatography of lysates of HHV-6B-infected mononuclear cells.

The herpes simplex virus UL20 protein compensates for the differential disruption of exocytosis of virions and viral membrane glycoproteins associated with fragmentation of the Golgi apparatus.

The Golgi apparatus is fragmented and dispersed in Vero cells but not in human 143TK- cells infected with wild-type herpes simplex virus 1. Moreover, a recombinant virus lacking the gene encoding the membrane protein UL20 (UL20- virus) accumulates in the space between the inner and outer nuclear membranes of Vero cells but is exported and spreads from cell to cell in 143TK- cell cultures. Here we report that in Vero cells infected with UL20- virus, the virion envelope glycoproteins were of the immature type, whereas the viral glycoproteins associated with cell membranes were fully processed up to the addition of sialic acid, a trans-Golgi function. Moreover, the amounts of viral glycoproteins accumulating in the plasma membranes were considerably smaller than those detected on the surface of Vero cells infected with wild-type virus. In contrast, the amounts of viral glycoproteins present on the plasma membranes of 143TK- cells infected with wild-type or UL20- virus were nearly identical. We conclude that (i) in Vero cells infected with UL20- virus the block in the export of virions is at the entry into the exocytic pathway, and a second block in the exocytosis of viral glycoproteins associated with cytoplasmic membranes is due to an impairment of transport beyond Golgi fragments containing trans-Golgi enzymes and not to a failure of the Golgi oligosaccharide-processing functions; (ii) these defects are manifested in cells in which the Golgi apparatus is fragmented; and (iii) the UL20 protein compensates for these defects by enabling transport to and from the fragmented Golgi apparatus.

Localization and putative function of the UL20 membrane protein in cells infected with herpes simplex virus 1.

The UL20 protein of herpes simplex virus 1, an intrinsic membrane protein, is required in infected Vero cells in which the Golgi apparatus is fragmented for the transport of virions from the space between the inner and outer nuclear membranes and for the transport of fully processed cell membrane-associated glycoproteins from the trans-Golgi to the plasma membrane. It is not required in the human 143TK- cell line, in which the Golgi apparatus remains intact. We report the following. (i) The UL20 protein was detected in infected cells beginning at 6 h postinfection and was regulated as a gamma 1 gene. (ii) Pulse-chase experiments revealed no detectable alteration in the mobility of the UL20 protein in polyacrylamide gels. (iii) In both infected Vero and infected 143TK- cells, the UL20 protein was detected by immunofluorescence in association with nuclear membranes and in the cytoplasm. Some of the cytoplasmic fluorescence colocalized with beta-COP, a protein associated with Golgi-derived transport vesicles. UL20 protein was present in virions purified from the extracellular space but could not be detected in the plasma membrane. These results are consistent with the hypothesis that UL20 is a component of virion envelopes and membranes of virion transport vesicles and is selectively retained from the latter in a Golgi compartment.

Polyvalent and monoclonal antibodies identify major immunogenic proteins specific for human herpesvirus 7-infected cells and have weak cross-reactivity with human herpesvirus 6.

Hyperimmune rabbit and mouse sera raised to human herpesvirus 7 (HHV-7)-infected cells and an immune human serum identified 20[35S]methionine-[35S]cysteine-labelled proteins specific for HHV-7-infected cord blood mononuclear cells, ranging in apparent M(r) from 136K to 30K. The major proteins had apparent M(r) values of 121K, 100K, 87K, 85K, 60K, 51K, 46K, 42K, 40K and 36K. The human serum also identified seven [3H]glucosamine-labelled glycoproteins, with apparent M(r) values of 100K, 89K, 82K, 67K, 63K, 53K and 41K. Four monoclonal antibodies (MAbs) specific for HHV-7-infected cells were derived. Two reacted with a family of five antigenically related polypeptides (87K, 85K, 70K, 61K and 57K in apparent M(r)), designated as the p85 complex. Two reacted with 121K and 51K M(r) proteins designated as p121 and p51, respectively. Human sera react with high frequency with the p85 complex and to a lesser extent with p121; hence these two proteins appear to be immunodominant for both humans and laboratory animals. The hyperimmune mouse serum and some of the MAbs showed some cross-reactivity with HHV-6A(U1102)- and 6B(Z29)-infected cells. The implications of cross-reactivity with respect to the human immune response to HHV-6 and -7 infections and prevalence analyses are discussed.

Herpes simplex virus (HSV) glycoprotein H is partially processed in a cell line that expresses the glycoprotein and fully processed in cells infected with deletion or ts mutants in the known HSV glycoproteins.

Cell lines that constitutively express herpes simplex virus 1 (HSV-1) glycoprotein H (gH-1) failed to synthesize the mature form of gH and accumulated a precursor-like form of the glycoprotein, which was retained intracellularly, most likely in RER. Fine-structure analysis of the oligosaccharides present in recombinant gH revealed oligosaccharides processed by RER enzymes; sialylated complex-type and biantennary oligosaccharides, which are assembled in the trans-Golgi, were absent. A small fraction had the characteristics of oligosaccharides processed by the early mannosidases of the Golgi. These findings suggest that a defect in the transport out of RER to the Golgi may account for the intracellular retention of the immature form of gH in cells that express the glycoprotein constitutively. Upon superinfection of cells expressing gH-1 with HSV-2, recombinant gH-1 underwent maturation, indicating that a viral function is required to attain full processing of gH. The known HSV glycoproteins do not appear to carry out this function, since in cells infected with deletion mutants in gD, gG, gE, and gE-gI, with a spontaneous gC- mutant, or with a temperature-sensitive mutant in gB, maturation of gH occurred independently of the presence or of the maturation of the single glycoproteins tested. The present findings together with previous observations on HSV, human CMV, and the EBV homologue of gH suggest that inability of gH to undergo full processing in the absence of viral protein(s) is a property of gH.

Glycoprotein D of herpes simplex virus encodes a domain which precludes penetration of cells expressing the glycoprotein by superinfecting herpes simplex virus.

Earlier studies have shown that herpes simplex viruses adsorb to but do not penetrate permissive baby hamster kidney clonal cell lines designated the BJ series and constitutively expressing the herpes simplex virus 1 (HSV-1) glycoprotein D (gD). To investigate the mechanism of the restriction, the following steps were done. First, wild-type HSV-1 strain F [HSV-1(F)] virus was passaged blindly serially on clonal line BJ-1 and mutant viruses [HSV-1(F)U] capable of penetration were selected. The DNA fragment capable of transferring the capacity to infect BJ cells by marker transfer contains the gD gene. The mutant gD, designated gDU, differed from wild-type gD only in the substitution of Leu-25 by proline. gDU reacted with monoclonal antibodies which neutralize virus and whose epitopes encompass known functional domains involved in virus entry into cells. It did not react with the monoclonal antibody AP7 previously shown to react with an epitope which includes Leu-25. Second, cell lines expressing gDU constitutively were constructed and cloned. Unlike the clonal cell lines constitutively expressing gD (e.g., the BJ cell line), those expressing gDU were infectable by both HSV-1(F) and HSV-1(F)U. Lastly, exposure of BJ cells to monoclonal antibody AP7 rendered the cells capable of being infected with HSV-1(F). The results indicate that (i) gD expresses a specific function, determined by sequences at or around Leu-25, which blocks entry of virus into cells synthesizing gD, (ii) the gD which blocks penetration by superinfecting virus is located in the plasma membrane, (iii) the target of the restriction to penetration is the identical domain of the gD molecule contained in the envelope of the superinfecting virus, and (iv) the molecular basis of the restriction does not involve competition for a host protein involved in entry, as was previously thought.

Glycoprotein C-dependent attachment of herpes simplex virus to susceptible cells leading to productive infection.

Herpes simplex viruses encode several glycoproteins dispensable for infection and replication in cell culture. Evidence is presented that there exist at least two pathways for viral attachment to cells, i.e., one mediated by the dispensable glycoprotein C (gC) and one independent of that glycoprotein. Thus, whereas the polycations neomycin and polylysine inhibit attachment but not entry of already attached herpes simplex virus 1 (HSV-1) into baby hamster kidney (BHK) cell line, they have no effect on HSV-2 attachment to the same cells (N. Langeland, H. Holmsen, G.R. Lilehaug, and L. Haarr, 1987, J. Virol. 61, 3388-3393; N. Langeland, L.J. Moore, H. Holmsen, and L. Haarr, 1988, J. Gen. Virol. 69, 1137-1145). We report that (i) analyses of intertypic HSV-1 X HSV-2 recombinants indicated that the HSV-2 locus which confers ability to infect BHK cells in the presence of neomycin or polylysine comaps with the gene specifying gC but not with or near the genes specifying the other viral glycoproteins (gB, gD, gE, and gG, and gI), (ii) the smallest HSV-2 DNA fragment capable of transferring this function to HSV-1 was a 2880-bp Sa/l fragment encoding the entire gC (UL44 open reading frame) gene, 515 bp of coding sequences from the UL43 open reading frame and 393 bp of coding sequences from the UL45 open reading frame, but analyses of the recombinant virus DNA excluded UL43 and most of the UL45 sequences, and (iii) definitive evidence that HSV-2 gC confers upon HSV the capacity to infect BHK cells in the presence neomycin or polylysine emerged from studies showing that site-specific mutagenesis which inactivated the gene yielded a recombinant whose attachment to BHK cells was blocked by the polycations. We conclude that in BHK cells there exists in addition to the pathway blocked by neomycina and polylysine a pathway which is parallel and HSV-2 gC dependent.

Herpes simplex virus glycoprotein D is sufficient to induce spontaneous pH-independent fusion in a cell line that constitutively expresses the glycoprotein.

Spontaneous small polykaryocytes were detected in a cell line designated BJ-o that harbors the BamHI J fragment of herpes simplex virus 1 DNA and expresses constitutively glycoprotein D (gD). The fusion activity of BJ-o cells correlated with gD production and was drastically reduced following exposure of the cells to monoclonal antibody HD1 to gD. Studies on the characteristics and requirements of cell fusion dependent on gD led to the conclusion that the characteristics and requirements for gD-mediated fusion activity of BJ-o cells are similar to those previously reported for cell fusion induced by the virus in that (i) polykaryocytosis was not augmented by exposure to medium of low pH with or without prior exposure to trypsin, (ii) the number of polykaryocytes was reduced following removal of terminal sialic acid residues by neuraminidase, and (iii) the number of polykaryocytes was augmented by masking of high-mannose N-linked oligosaccharides with concanavalin A or with its reduced form, succinyl concanavalin A. This effect was reversed by competition with mannose.

Individual herpes simplex virus 1 glycoproteins display characteristic rates of maturation from precursor to mature form both in infected cells and in cells that constitutively express the glycoproteins.

Pulse-chase experiments in conjunction with quantitative immunoprecipitation have been used to study the time-course of conversion from precursor to mature form of herpes simplex virus 1 glycoproteins C, D and B (gC, gD, and gB). The experimental systems employed were two infected cell lines and cells that constitutively express gD or gB. The relative rates of conversion among the glycoproteins did not vary in the systems used; the rate of maturation of gC was about two-fold higher than that of gD which, in turn, was about one and a half-fold higher than that of gB. Treatment with phosphonoacetate which inhibits viral DNA synthesis and hence virion morphogenesis induced a striking increase in the time course of conversion of immature gC, gD, and gB to fully glycosylated forms when measured late in the infection. The model of HSV glycoproteins maturation as integral components of the virion envelope is discussed.

Comparative biochemistry of the ubiquinol-cytochrome c oxidoreductase (EC 1.10.2.2) isolated from different heart mitochondria.

The ubiquinol-cytochrome c oxidoreductase (bc1 complex, EC 1.10.2.2) has been isolated from the heart mitochondria of beef, chicken, turkey, duck and tuna with an identical procedure. The polypeptide composition of the different complexes, compared using SDS-polyacrylamide gel electrophoresis, shows that the three subunits carrying the prosthetic groups of the enzyme are highly conserved in all species. Also the large subunits I and II (core proteins) and band VI appear to be conserved in structure, while subunits VII and VIIa show a most remarkable structural variation in the various complexes. The steady-state ubiquinol-cytochrome c reductase analysis of the active enzymes indicates that all the bc1 complexes follow essentially a ping-pong mechanism, with the cytochrome c substrate displaying a partial competitive inhibition vs the ubiquinol substrate. The cytochrome c specificity of the reductase activity clearly is different in the various bc1 complexes, whereas the quinol specificity appears to be identical in all the enzymes.