- Volume 66, Issue 9, 1985

Moloney murine sarcoma virus (Mo-MuSV) was one of the most widely studied mammalian retroviruses because it had acquired the cellular sequence called mos (Van Beveren et al., 1981a; Frankel & Fischinger, 1976), one of the first oncogenes identified. But difficulties with the low level of expression of the viral mos (v-mos) protein and apparent lack of cellular mos (c-mos) expression have decreased interest in Mo-MuSV. However, a variant of Mo-MuSV derived by mutagenesis and biological selection, ts110, clearly produces higher levels of a v-mos protein. The purpose of this review is to summarize important findings and properties of this system. Briefly, gag-mos proteins are produced in easily detectable amounts in cells infected with ts110 Mo-MuSV or in wild-type revertants derived from ts110 virus-infected cells. The ts110 mutant virus exhibits two conditional defects. One defect affects the production of the mRNA for the gag-mos protein and the other affects the stability of the gag-mos protein and its associated kinase function.

The number and size of the major virion polypeptides have been determined by SDS–PAGE for the 22 Staphylococcus aureus phages of the International Typing Set, plus phages 11 and 80α. Virion ultrastructure was examined by electron microscopy after negative staining with ammonium molybdate. In addition, serogroup B phages were disrupted and fractionated into head, tail-tube and baseplate components and major polypeptides assigned to these substructures. The number and size of the polypeptides correlated closely with the division of aureophages into four serogroups (A, B, F, L), although serogroup L was represented in the set by only a single phage (187). Apart from serogroup B, however, the polypeptide patterns did not reflect differences between lytic groups. Within serogroup B, polypeptide analysis yielded characteristic patterns for lysogroups I, II and III. Ultrastructural analyses confirm the data provided by polypeptide analysis. Thus, phages from the four serogroups can be identified on the basis of tail-tube length alone, although the differences between phage 187 and members of lysogroups I and III in serogroup B were less than 20 nm, or approximately 12% of the total length. Serogroup A virions differed from those of the other serogroups in that all members of the typing set in this group had elongate, rather than isometric, heads.

The in vitro transcription pattern of BglII-digested ø3T DNA is described. Eight BglII fragments that hybridized to in vitro transcription products were unequivocally identified. A further hybridizing region corresponding to a BglII triplet was also revealed, giving a total of nine to 11 BglII fragments. These represent 47 to 53% of the ø3T genome. Transcription was shown to initiate within BglII fragments B, G, C, H, I, F and J, implying that all of these contain at least one promoter. The relevance of these data to the construction of a cloning vector based on ø3T is discussed.

Hybrid cell lines were prepared by the fusion of mouse myeloma cells with the spleen cells of Wistar-Furth rats that had been immunized with a Moloney sarcoma virus (Mo-MuSV)-induced tumour, MFU. Two immunization protocols were designed. In the first, the animals received several injections of irradiated (10000 rad) cells of a tumour cell line established in vitro, MFU-67. The rats received a booster injection 3 days prior to fusion. In the second protocol, immunization was the result of simple tumour growth, and no booster was given. Hybrids were tested by immunofluorescence for the production of immunoglobulins reacting with mouse cells acutely infected with Mo-MuSV. Over 20% of reactive hybrids were observed in the tumour growth protocol, and about 10% in the irradiated cell protocol when the last injection of the series was given 2 weeks before fusion. After 6 months, the proportion fell to 3%. Hybrid lines producing antibody to p30, the major core polypeptide of murine retroviruses, were obtained by cloning. Three of these were selected for closer study and were found to recognize three non-overlapping epitopes on p30. By direct and competitive binding in ELISA tests, the three epitopes were found to have very different distribution patterns among the various strains and isolates of murine retroviruses.

Monoclonal antibodies were raised against a cell surface glycoprotein (46K antigen) expressed by a Moloney sarcoma virus (Mo-MSV)-transformed STU mouse cell line (Sac). Although non-productively transformed Sac cells expressed the 46K antigen at their surface, transplantation of Sac non-producer cells into immunocompetent mice failed to induce anti-46K antibody production. Only transplantation of helper virus-superinfected Sac cells led to the development of anti-46K antibodies in addition to antibodies against helper virus structural antigens. Hybridomas producing anti-46K antibodies were established by fusion of mouse myeloma cells with spleen cells taken from mice bearing Sac tumour cells infected with Moloney helper virus. The monoclonal antibodies were subsequently tested against STU mouse transformants [spontaneously transformed or experimentally transformed with the progressor strain of Mo-MSV and the chemical carcinogen 3-methylcholanthrene (MCA), respectively] and BALB/c mouse transformants [transformed with Mo-MSV, simian virus 40 (SV40) or MCA]. Only one of the transformed cell lines tested, a non-producer transformant (PV-TC-77) induced with the progressor strain of Mo-MSV in an STU mouse was found to express a 46K antigen on the cell surface. The 46K surface antigen expressed by PV-TC-77 cells exhibited the same serological, biochemical and biological properties as the 46K Sac cell surface antigen. Though detectable on the surface of PV-TC-77 non-producer cells, immunocompetent syngeneic recipients of non-producer PV-TC-77 cells remained unresponsive against their 46K surface antigen. Only transplantation of helper virus-infected PV-TC-77 producer cells led to antibody development against the 46K surface antigen.

An adenovirus (Ad) interserotypic recombinant (H2cyt141) between temperature-sensitive mutant H2ts111 of Ad2 and deletion mutant H5dl313 of Ad5 was isolated and characterized. It was phenotypically ts +, dl +, hr + and formed large plaques (or cytocidal: cyt). It contained the right 89% of Ad5 DNA and the leftmost 11% of Ad2 DNA. Genetic recombination data suggested the cytocidal mutation lay in the transforming region E1B, confirming sequence analysis. The cytocidal effect resulted in part from the breakdown of cellular DNA. Host cell and virus DNA breakdown induced by H2cyt141 appeared cell-dependent: it occurred in HeLa, KB or BHK-21 cells, but not in CV1 or 293 cells. In human cells the cyt effect was recessive and adenovirus DNA degradation was prevented by co-infection with adenovirus wild-type (H2WT), other adenovirus serotypes or simian virus 40 (SV40). In simian cells, H2cyt141 did not inhibit SV40 DNA replication, unlike H2WT. The amount of H2cyt141 DNA integrated in human cell DNA at early stages of the lytic cycle was found to be significantly lower than for H2WT. Novobiocin inhibited viral DNA breakdown in human cells. Cellular DNA extracted from H2cyt141-infected cells exhibited a repeat band pattern in gel electrophoresis reminiscent of the nuclease digestion pattern of chromatin, with monosome-size fragments as the digestion limit. The H2cyt141-induced nucleolytic effect would therefore occur in the linker regions of cell DNA and might result from the observed stimulation (by a factor of greater than 100) of an acidic (optimum pH 4.0) endonuclease activity. The nucleolytic effect also appeared to be recessive in vitro and absent in mixed samples containing extracts from H2cyt141-infected cells plus extracts from H2WT- or mock-infected cells. The virus gene product responsible for the enhancement of the acidic endonuclease was found to function stoichiometrically and not catalytically. The cytocidal and nucleolytic effects of the viral E1B region 19K protein may be mediated by a cellular inhibitor of acidic endonuclease.

The major structural polypeptide of rotaviruses is p45K (VP6), which forms the morphological subunit of the inner capsid. Such subunits show a trimeric structure when examined with the electron microscope. Treatment of single-capsid rotavirus particles with 1.5 m-CaCl2 removes p45K, resulting in the generation of smooth cores. Sucrose density gradient centrifugation analysis of the removed p45K revealed that it has a sedimentation coefficient close to 7.3S, compatible with an oligomeric (possibly trimeric) structure. Polyacrylamide gel electrophoresis under reducing or non-reducing conditions indicated that p45K has intramolecular but not intermolecular disulphide bonds, suggesting that interactions between p45K monomers may be due to some other type of association, such as hydrophobic or charge interactions. Velocity sedimentation of infected cell extracts revealed that native p45K also behaves as an oligomeric protein. Such results were confirmed using p45K partially purified by DEAE-cellulose chromatography. The evidence obtained indicated that all p45K present in the virion is in the oligomeric form, not associated by disulphide bonding, and that most native p45K present in the infected cells is also in the oligomeric form, probably as a consequence of early protein-protein interaction in rotavirus morphogenesis.

Evidence of multiple genotypes of bluetongue virus (BTV) serotype 11 simultaneously infecting individual cattle was demonstrated in a California sentinel herd naturally infected with two BTV serotypes (11 and 17). Monitoring of weekly virus isolates by PAGE demonstrated genome segment diversity among BTV serotype 11 isolates and an individual bull simultaneously infected with multiple electropherotypes of this serotype. No electrophoretic variations were apparent in BTV serotype 17 isolates. The results of this study indicate that multiple plaque clonings and comparative electrophoresis are required to analyse BTV field isolates accurately for the presence of mixed infections.

Two transmissible gastroenteritis (TGE) virus mutants (188-SG and 152-SG) were obtained from a low-passage virus strain (D-52) by 188 and 152 cycles of stomach juice treatment and multiplication in cell culture. Compared to the high-passage Purdue-115 and the original D-52 strains, these mutants were more stable at pH 2.0, more resistant to pepsin and trypsin, and characterized by a small plaque phenotype. In vivo, the two mutants were not found to be virulent for 4-day-old piglets and sows after oral inoculation. To test induction of lactogenic immunity, the 188-SG mutant was administered orally to pregnant sows (6 or 7 weeks before parturition) followed by one intramuscular booster (1 week before parturition). After challenge with virulent TGE virus, piglet mortality 7 days after exposure was reduced (to 22%) as compared to the death rate in piglets from control sows (91%).

Sodium bisulphite modification of foot-and-mouth disease virus (FMDV) RNA in solution indicates that the majority of the poly(C) tract in the RNA is single-stranded in concordance with previous results with encephalomyocarditis virus RNA. The reaction kinetics are biphasic; 60% of the cytidylic acid in the poly(C) tract reacts like synthetic poly(C), and the remainder with the kinetics of the cytidylic acid in the rest of the RNA. The reactivity of the poly(C) tract with poly(I) indicates that it is looped out and exposed in the RNA. The deamination reaction has also been used to investigate the structure of the replicative form (RF) and replicative intermediate (RI) isolated from infected cells. Analysis by gel electrophoresis of the long RNase A- and T1-resistant oligonucleotides of RI suggests that it has five single-stranded poly(C) tracts to every one which is base-paired. Bisulphite reactivity of the poly(C) tract and gel electrophoresis of the ribonuclease-resistant oligonucleotides of RF indicate that the poly(C) is base-paired to a poly(G) tract in this molecule. The presence of a poly(G) tract in RF and RI provides unequivocal evidence that the poly(C) is replicated via poly(G) in the negative strand.

The episomal copies of Epstein-Barr virus (EBV) DNA are in a chromatin structure in the lymphoblastoid cell line B95-8. Nucleosomes on EBV DNA have the same spacing as those in cellular chromatin. Some of the EBV DNA is not in nucleosomes; this probably corresponds to DNA which is being packaged. Several DNase hypersensitive sites have been mapped on the EBV genome and patterns of CpG methylation are examined.

Herpesvirus Movar 33/63 is the prototype strain of a group of slow growth bovine herpesviruses which have been reported to exhibit cytomegalovirus-like characteristics. These viruses have the ability to produce long-term persistent infections of spleen and other lymphoreticular organs in both cattle and rabbits. Rabbits were inoculated with a suspension of Movar 33/63 propagated in cell culture, and sacrificed at intervals between 3 days and 49 weeks post-infection. Cell-free infectious virus was detected only in conjunctival secretions, buffy coat and spleen homogenates up to 7 days post-infection. Beyond this brief acute replication period, co-cultivation or explantation was required for the detection of viral infectivity. The spleen, the only organ from which virus was consistently recovered, exhibited the highest infectious titres as detected by infectious centre assay. The use of several cell isolation techniques (including solid-phase fractionation on ligand-coated surfaces, nylon wool filtration, affinity chromatography, immunocytolysis and plastic surface adherence) allowed separation of B-enriched, T-enriched and non-T, non-B cell fractions. Infectivity during the acute and persistent phase of the infection was associated with the non-T, non-B population which was highly enriched in adherent and non-adherent spleen mononuclear phagocytes. No virus was isolated from either T or B cells.

Xanthates have recently been shown to inhibit the replication of both DNA and RNA viruses in vitro. The antiviral activity was exerted only under acidic pH conditions. Curative effects in vivo on herpes simplex virus (HSV)-induced skin lesions were only observed when the xanthate compound was administered in the form of an ointment containing acidic buffer (sodium phosphate pH 5.0). Advanced HSV-2-induced skin lesions in mice were healed by topical treatment with the xanthate compound. HSV-1-induced lesions on skin of guinea-pigs were cured within 2 days even when the treatment was initiated as late as 4 days after infection. Both HSV-1 DNA synthesis and virus production in the skin of guinea-pigs were also shown to be inhibited after treatment with the xanthate compound.

High titres of rat interferon (IFN) can be produced by rat embryo fibroblast (CD) cells after treatment with Newcastle disease virus. This CD IFN was characterized by SDS–PAGE, and was found to contain three species at 30K to 33K, 25K to 27K and 17K to 22K mol. wt. Antibody affinity chromatography revealed that 95% of the CD IFN bound to an anti-human leukocyte IFN antibody column, 34% to an anti-mouse L cell IFN antibody column and none to an anti-human fibroblast IFN antibody column. The IFN that bound to the anti-human leukocyte IFN antibody column contained all three molecular weight species of IFN. However, the material bound to the anti-mouse L cell IFN antibody column only showed IFN activity of the 30K and 20K species. Examination of the heterologous antiviral activity of the unseparated CD IFN and of the IFN separated by antibody affinity chromatography revealed the same patterns of activity: 35 to 50% of homologous activity on guinea-pig cells, 10 to 20% on mouse cells, 2 to 4% on human cells and none on bovine cells. The data suggest that a major portion of this rat fibroblast IFN is related to alpha IFNs produced by other species.

The mode of entry of West Nile virus (WNV) into the macrophage-like cell line P388D1 was investigated at the electron microscopical level using synchronized infections. The presence of the antiviral monoclonal antibody F6/16A at a concentration that enhanced viral attachment to P388D1 cells ninefold made no difference to the entry pathway of WNV. In both the absence and presence of F6/16A the initial uptake of single viral particles was mediated by coated pits, and started within 30 s of warming the cells to 37°C. Viral particles later appeared in fully or partially coated vesicles and later in uncoated prelysosomal endocytic vacuoles before degradation in lysosomes. However, aggregates of viral particles (five or more virus particles in cross-section), appeared to be phagocytosed whole by cells in a process which involved aggregates being engulfed by extensions of the plasma membrane. This process exhibited a slower time course than the uptake of single viral particles, becoming prominent 15 to 30 min after warming the cells to 37°C. The involvement of a prelysosomal vacuolar compartment in the entry process was shown by a failure to stain for acid phosphatase. This compartment could be specifically loaded with viral particles when viral internalization occurred at 20°C in the presence of 50 mm-ammonium chloride.

The synthesis of the two respiratory syncytial (RS) virus glycoproteins (VP66 and VP84) was examined under standard conditions and after treatment with tunicamycin and monensin. The protein backbone for VP66, the fusion protein (F1,2) is co-translationally glycosylated to form F0, which is cleaved to form F1,2 by 20 min of chase. Monensin treatment inhibited the cleavage of F0 over an 80 min chase period, indicating that this occurred late in the transit of F0 through the Golgi apparatus or after exit from the Golgi apparatus. Tunicamycin treatment resulted in the synthesis of a 50K to 55K unglycosylated F0 which is cleaved to a 40K protein. VP84, the large glycoprotein, contains a protein backbone of only 26K to 30K which is modified by N-linked and probable O-linked glycosylation. Tunicamycin treatment results in the synthesis of a 70K protein (p70) which incorporates [3H]glucosamine and [3H]fucose but not [3H]mannose. Glycosylated precursors varying in mol. wt. from 29K to 45K (p45) are found in infected cells at regular 2K to 3K intervals, producing a ‘ladder’ effect. The step from p45 to VP84 is severely delayed by monensin treatment thereby enhancing the ‘ladder’ effect of the precursors.

Monospecific rabbit antisera against purified herpes simplex virus type 1 (HSV-1) gB, gC or gD antigens or polyvalent rabbit antiserum against equine herpesvirus type 1 (EHV-1)-infected cells was used in Western blotting to identify antigenically related proteins in cells infected with HSV-1, HSV-2, bovine mammillitis virus or EHV-1. Monomeric and oligomeric polypeptides related to HSV-1 gB were found in cells infected with each of the four herpesviruses. The gC antiserum was specific for HSV-1 and the gD antiserum reacted only with polypeptides in HSV-1- or HSV-2-infected cells. Neither gC nor gD antiserum revealed the existence of oligomeric forms of the glycoproteins with the single exception of HSV-1 strain KOS which had a heat-unstable high molecular weight form of gD. The EHV-1 antiserum reacted with high molecular weight polypeptides of the same mobility as the oligomeric form of gB in cells infected with each of the four herpesviruses.

The characteristics of four human cytomegalovirus (HCMV)-specific monoclonal antibodies as assessed by ELISA, immunofluorescence, immunoprecipitation and Western blotting are described. Two antibodies recognized a 67K late polypeptide of HCMV, one recognized 43K and 79K polypeptides present early and late in HCMV-infected cells, and the fourth identified a 72K early nuclear protein of HCMV. The antibodies recognized these antigens in all HCMV isolates tested by immunofluorescence and ELISA, but demonstrated inter-isolate variations in polypeptides recognized by Western blotting.

Chicken sera may contain components that immunologically crossreact with avian retroviral glycoprotein. Sera from chickens carrying the endogenous viral loci ev3 and ev6 contain glycoproteins with molecular weights of approx. 85000, which on the basis of tryptic peptide analysis are related to exogenous viral glycoprotein. Such endogenous viral glycoproteins are present in a free form, lacking disulphide-bonded gp35.

The extent of homology between the translation products of the HZ2 strain of feline sarcoma virus (HZ2-FeSV) and the Abelson murine leukaemia virus (A-MuLV) was examined immunologically and biochemically. Antiserum prepared against the v-abl-encoded determinants of the A-MuLV polyprotein P120gag-abl was also found to precipitate specifically the 98K mol. wt. HZ2-FeSV protein (P98gag-abl). The basis for this immunological crossreactivity was indicated by the findings that the two proteins had at least six [35S]methionine-containing tryptic peptides and at least eight [35S]methionine-containing chymotryptic peptides in common. Each of the two proteins also had tryptic and chymotryptic peptides which were unique. Both proteins were associated with tyrosyl kinase activities which exhibited some similar biochemical properties in vitro. However, the HZ2-FeSV-associated activity was much more sensitive to competitive inhibition by nucleoside and deoxynucleoside diphosphates than was the A-MuLV-associated activity. These results suggest that, while the gag-abl translation products of these two independent isolates of transforming retrovirus are highly related structurally and functionally, the differences in structure contribute to differences in enzyme activity. Further comparative studies of these two proteins should play an important role in determining their roles in induction of two different types of malignancy: lymphosarcoma in the case of the A-MuLV protein and fibrosarcoma in the case of the HZ2-FeSV protein.