- Volume 46, Issue 2, 1980

Rauscher leukaemia virus (RLV), extensively purified by rate and density gradient centrifugation procedures, exhibited aminopeptidase (AP) activity. The amount of virion activity was about 0.005 times the specific activity of purified hog kidney aminopeptidase M (EC 3.4.11.2). The activity was also found in purified Moloney and Gross leukaemia viruses, but not in comparable gradient fractions of uninfected JLSV-9 cells. Furthermore replacement of serum by bovine serum albumin in the growth medium of virus producing cells did not affect the AP activity. These results indicate that murine leukaemia viruses (MuLV) possess a tightly bound AP activity which is present as a minor component of the virion preparation and is probably not due to host cell membrane or serum contamination. Characterization of the pH optimum and substrate specificity show the MuLV aminopeptidase activity is similar to but different from both hog kidney, leucine aminopeptidase (EC 3.4.11.1) and aminopeptidase M (EC 3.4.11.2).

Peritoneal macrophages from mice infected with herpes simplex virus type 2 (HSV-2) exhibited extrinsic antiviral resistance. When the macrophages were cocultivated in vitro with virus-infected cells the yield of virus was reduced markedly. Activity was not present 1 to 2 days p.i., peaked at 3 to 4 days, declined by 7 days and was absent at 14 days after HSV-2 infection. The extrinsic antiviral activity was limited to the adherent peritoneal macrophage population. The macrophage antiviral activity was also dose-dependent, with approx. 106 macrophages (macrophage:host cell ratio of approx. 2:1) reducing virus plaques by > 90% and virus yield 1.5 to 3.0 log10. Comparable extrinsic antiviral activity was also exhibited by Corynebacterium parvum- or thioglycollate-elicited peritoneal macrophages. The macrophage activity was not species-specific, activity on Vero cells or syngeneic mouse embryo fibroblasts being comparable. Activity was also not virus-specific, as the active macrophages also inhibited vesicular stomatitis virus (VSV). The antiviral effects required viable macrophages; cell lysates did not inhibit virus growth.

A temperature-sensitive (ts) strain of tobacco mosaic virus (TMV), Ls1, was further investigated with regard to its behaviour at permissive (22 °C) and nonpermissive (32 °C) temperatures at the primary infection sites in tomato leaf epidermis. Tomato leaflets were inoculated with Ls1 or a wild-type strain, L, cultured at 22 or 32 °C and the epidermis isolated; virus spread was followed by staining with fluorescent antibody after enzymic digestion of the upper cell wall. Profiles of epidermis stained with fluorescent antibody indicated that Ls1 multiplies in primary-infected cells but that it cannot move to the neighbouring cells at the nonpermissive temperature.

A double antibody competitive binding radioimmunoassay (RIA) was developed as a tool for investigating the involvement of measles virus in persistent virus infections. The assay employs 125I-labelled measles virus nucleocapsids as the labelled antigen. Nucleocapsids were purified from cytoplasmic extracts of virus-infected Vero cells, treated with trypsin to prevent clumping and iodinated to a specific activity of about 15 µCi/µg. Electrophoretic analysis of iodinated trypsintreated nucleocapsids revealed only labelled polypeptide with a relative mol. wt. (Mr) 40000, indicating that trypsin had cleaved the 60000 mol. wt. native polypeptide to a 40000 mol. wt. subunit. The assay could detect as little as 0.1 ng of nucleocapsid protein. Either native (Mr 60000) or cleaved (Mr 40000) nucleocapsid polypeptide was detected in the assay. As much as 100 µg of protein from uninfected Vero cells did not react in the RIA. Another measure of specificity was the fact that 400 ng of purified nucleocapsid protein from simian virus 5, Newcastle disease virus or canine distemper virus did not react in the assay. In the RIA, nucleocapsid antigen of virus isolated from subacute sclerosing panencephalitis (SSPE) was indistinguishable from that of Edmonston strain measles virus, indicating antigenic homology between the 40000 mol. wt. nucleocapsid polypeptide of the SSPE virus and that of measles virus.

In human KB cells productively infected with adenovirus 2 (Ad2) high mol. wt. forms of DNA have been described that contain virus DNA integrated into cellular DNA. The implications of this discovery are not yet understood. We studied the competence for the formation of such DNA forms of cell species closely related with respect to their origin, although diverse with respect to their proliferative capacity. Eight human and simian cell species infected with three adenovirus strains, representing adenovirus subgroups of different oncogenic potential, were included in the study. The cell systems comprised primary cells of various tissue origin, semi-permanent cells and cell lines originally derived from carcinoma cells. A DNA species was recognized as virus high mol. wt. DNA by the criteria of its high sedimentation value in alkaline sucrose gradients, ability to hybridize to virus DNA, a buoyant density intermediate to that of virus DNA and cellular DNA and the ability to liberate DNA with the density of virus DNA upon ultrasonic fragmentation. The results indicate that formation of the high mol. wt. DNA is not correlated with the state of transformation of the cell nor with the oncogenic potential of the infecting adenovirus strain and could be demonstrated in all virus cell combinations tested. Thus integration of virus sequences into the cellular genome appears to be a common event in productive infection of many adenovirus-cell systems, though cellular as well as virus factors seem to exert modulating influences.

The antiviral responses in mice to intranasal inoculation with Sendai virus are described. To investigate the relative importance of the humoral, cell-mediated and interferon responses, the pathogenesis of this infection was studied in animals which were immunocompetent, T cell-deprived or immunosuppressed with cyclophosphamide. Treatment with cyclophosphamide converted the mild, self-limiting infection observed in immunocompetent mice into a severe and frequently lethal pneumonic disease. This was associated with an enhanced interferon response but not detectable antibody or cell-mediated immune response. T cell-deprived mice suffer an infection of intermediate severity associated with an increased interferon response, a normal humoral immune response and no cell-mediated immune response. The implications of these results in relation to the role of the antiviral responses in recovery from Sendai virus infection are discussed.

The indirect immunoperoxidase technique was used to investigate the development of the virus-specific intracellular and cell membrane antigens in cells infected with the Shope fibroma virus. Starting with 6 h p.i., virus antigen formed distinct inclusions within the cytoplasm frequently enclosed by endoplasmic reticulum. The endoplasmic reticulum disappeared almost completely 10 to 12 h p.i., coincidentally with the beginning of virus formation. The virus antigen was distributed throughout the cytoplasm. At the same time virus-induced antigen began to appear at the cell membrane and subsequently increased. No cytochemical staining could be observed on the endoplasmic reticulum, within the nucleus and within immature and mature virus particles. The correlation between antigen synthesis and changes in cell ultrastructure is discussed.

Tick-borne encephalitis (TBE) virus was crosslinked by dimethylsuberimidate (DMS) and the cleavable dimethyl 3,3′-dithiobispropionimidate (DTBP). Analysis by SDS–PAGE revealed polymers of the virus core protein V2 and the glycoprotein V3 in continuously decreasing amounts. The formation of higher order complexes was not favoured over the formation of lower order complexes. This is consistent with an even distribution of V3 molecules on the surface of the virion and of V2 in the core. The formation of polymers was completely abolished by SDS, whereas crosslinking of TBE virus disrupted with a large excess of mild detergents (Triton X-100, octylglucoside, Na-deoxycholate) still yielded V3 dimers but only negligible amounts of higher polymers. This indicates that in the presence of these detergents the basic subunit of the TBE virus envelope is composed of two V3 molocules, probably associated with V1. Using two-dimensional PAGE analysis of DTBP crosslinked complete virions or cores, no heterocomplexes between different virus proteins could be found which were small enough to penetrate a 5% gel. Crosslinking between V3 molecules only and V2 molecules only was therefore highly favoured over other reactions.

Fresh human sera neutralize Friend leukaemia virus (FLV). The activity is abolished by heating (56 °C for 30 min), hydrazine and EDTA treatment, suggesting the involvement of complement. Human sera also contain antibody which specifically reacts with FLV. Following incubation with human whole serum, IgG and F(ab′)2 fragments, FLV acquire the ability to bind anti-human Ig sera, as shown in neutralization and radioimmunoprecipitation assays. The same is true for FLV-infected cells but not for uninfected control cells, as assessed by immunofluorescence. The binding of antibody to FLV-infected cells is prevented by pre-treating the cells with antisera to FLV and FLV-gp71, and is competed by FLV, FLV-gp71 and, to a lesser extent, by FLV-p12 and p30.

Heat-resistant derivatives of a Bacillus subtilis 168 strain carrying an xhi mutation, which causes heat-sensitive induction of the PBSX prophage, have been isolated and screened for the acquisition of auxotrophy. Two classes of auxotrophs were isolated, namely Pro− and Pro−Met−; they lacked the ability to produce PBSX, as shown by their resistance to mitomycin C-induced lysis. The proline and methionine requirements and the resistance to mitomycin C were shown to segregate together in phage PBS1-mediated transduction crosses and to be linked to thiB, which is known to be co-transducible with the PBSX prophage. It was therefore proposed that these strains had deletions which removed all or part of the PBSX prophage together with adjacent bacterial DNA encoding the pro(AB) and metC genes. The met mutation was shown to be metC in PBS1 transduction crosses; this gene is known to be co-transducible with the PBSX prophage. The proline requirement was probably due to the deletion of a pro gene which was demonstrated to lie between the PBSX prophage and metC and which was 90% co-transducible with metC.

These deletions have been transduced into a strain which was cured of phage SPβ, another bacteriophage carried by B. subtilis 168. No phage particles could be seen in mitomycin C-induced cultures of such strains.

The PBSX-deletion strains grew with the same generation time as the PBSX+ parent in L-broth (27 min at 35 °C) but they were slower in minimal medium (e.g. 72 min as against 51 min in the PBSX+ strain). Besides being resistant to mitomycin C-induced lysis, the deletion strains were also resistant to lysis induced by thymine starvation of thymine auxotrophs and the loss of viability of these strains after thymine starvation was 100-fold less than in the PBSX+ parent. The deletion strains had not, however, lost the bacterial autolytic enzymes, since they were still susceptible to lysis when placed under semi-anaerobic conditions.

In wheat germ extracts and reticulocyte lysates the genome RNA molecules of tomato black ring virus (TBRV), RNA-1 (mol. wt. 2.8 × 106) and RNA-2 (mol. wt. 1.6 × 106), were translated into products of maximum mol. wt. 2.2 × 105 and 1.6 × 105, respectively. These products represent about 80% and 100% of the coding capacity of the two RNA species. The 1.6 × 105 mol. wt. polypeptide reacted with antiserum to TBRV particles but the translation products of RNA-1 did not; this is additional evidence that RNA-2 contains the coat protein cistron.

Satellite RNA molecules (RNA-3, mol. wt. 4.8 × 105) associated with strain S of TBRV, like those associated with strain G, were translated in wheat germ extracts into a polypeptide of mol. wt. 4.8 × 104; this did not react with TBRV antiserum. Protease digestion released peptides from the translation product of strain S RNA-3 which were different from those released from the translation product of strain G RNA-3, suggesting that the two kinds of satellite RNA molecules differ in base sequence.

The sedimentation coefficients of tobacco necrosis virus strain D (TNV d ) in sodium phosphate at pH 5 and pH 7 are 104S and 97S respectively. The change in sedimentation coefficient between pH 5 and 7 is fully reversible and the decrease on transfer to pH 7 is unaffected by the presence of Mg2+. TNV d is disrupted by Bacillus subtilis protease at pH 7.5 but not at pH 5; ribonuclease neither disrupts virions nor destroys the infectivity of TNV d at either pH 7.5 or pH 5. Virions are markedly unstable at alkaline pH but the effectiveness with which different buffers dissociate TNV d varies. Virus is largely resistant to dissociation by 1 m-NaCl, 1 m-urea, 10 m-ethanediol and 1% sodium dodecyl sulphate (SDS) at or below pH 6 and is largely susceptible to dissociation by these agents at or above pH 7. Our results suggest that as the pH is lowered some non-covalent bonds important for virion stability may be formed and/or some electronic repellent interactions causing virion instability may be weakened.

In transformed mouse embryo cells, type II interferon had much less antiviral activity than type I interferon. In non-transformed cells, the two interferons had similar high activity. Reversal of the phenotype of Moloney sarcoma virus (MSV) transformed cells by sodium butyrate restored their sensitivity to the antiviral action of type II interferon. Additional evidence for a role of the cytoskeletal network in the action of type II interferon is that its antiviral effect is reduced by cytochalasin B, colchicine or vinblastine. MSV-transformed cells, selected for their resistance to the antiviral action of type I interferon, were sensitive to type II interferon. These differences in the effects of type I and II interferon on transformed cells are at present unexplained, but suggest that they have at least partially separate mechanisms of action.

G1-arrested BHK21 cells infected with adenovirus type 2 were studied to determine the effect of infection on host DNA synthesis in a productive cycle of infection. At various intervals following infection, analysis of the intracellular DNA was carried out by zonal sedimentation centrifugation through alkaline sucrose gradients. In addition to material which sedimented to the position of the virus marker (34S), a class of high mol. wt. DNA (40 to 100S) was found to be synthesized beginning approx. 13 h p.i. and continuing up to about 35 h p.i. DNA-DNA hybridization studies on this newly synthesized DNA showed it to be cellular DNA. When this material was density labelled with 5-bromodeoxyuridine and centrifuged to equilibrium through alkaline CsCl gradients, it was found to be the product of semi-conservative replication and not of repair synthesis.

The synthesis of polypeptides in infected Vero cells has been studied with two tissue culture adapted strains of mumps virus. Minor differences between the strains are found in the mobilities of the virus-specific polypeptides in SDS-PAGE. Eight virus-induced polypeptides have been identified with mol. wt. of 180K, 80K, 71K, 69K, 45K, 39K, 23K and 17K. These may correspond to the L, HN, N, F0, P, M, C and S polypeptides identified in other paramyxoviruses. The 69K F0 glycoprotein is probably a precursor for glycopolypeptides F1 and F2 with mol. wt. 61K and 14K, respectively.

Strains of yellow fever virus (YFV), Venezuelan equine encephalomyelitis virus (VEEV) and Semliki Forest virus (SFV) have been used to compare the stimulations of regulatory immunity (pre-challenge), antibody synthesis and protective immunity (post-challenge) in athymic-nude and normal mice. Similarly, direct assessments have been extended to athymic recipients of normal spleen cells and to adoptively immunized mice. The results indicate that the responses of mice to different togaviruses or strains of togaviruses may be differentially T-lymphocyte dependent at any one or more of the above three stages of host response. T-cell reconstitution or adoptive immunization may be effective only for the virus strains of highest immunogenicity. These results suggest a resolution of T-lymphocyte dependence at three levels of host response to virus infections. This approach may be of value in the similarly direct in vivo differentiation of other virus strains and as a practical framework for the consideration of the in vivo significance of the variety of in vitro lymphocyte markers.

Thirteen thermosensitive (ts) mutants of alfalfa mosaic virus (AMV), a virus with a tripartite genome, are described. Eight of these mutants were spontaneous, one was induced with HNO2 and four were induced by u.v. irradiation of one purified component. Using supplementation tests six ts mutations were located on top component b (Tb), three on middle component (M) and four on bottom component (B). Complementation tests with mutants with ts defects on the same component showed that none of the ts mutants on Tb could complement each other; the three ts mutants on M could be subdivided into two complementation groups, while only one pair of mutants showed complementation from the four ts mutants on B. It was demonstrated that the coat proteins from the six ts mutants on Tb were not able to activate the AMV genome at 30 °C in tobacco.

Polyhedral virus-like particles, approx. 39 nm in diam. (39-nm VLPs), have been studied by electron microscopy in thin sections of Lentinus edodes (Berk.) Sing. monokaryon. They were common in the cytoplasm and in vacuoles of the hyphal cells, either irregularly scattered or in aggregates which were sometimes crystalline especially in old mycelium. Crystals were found almost exclusively within cytoplasm and vacuoles in disintegrating cells. Examination of fused monokaryons suggests that the cytoplasmic 39 nm VLPs may move from one cell to another through the breakdown of a dolipore septum during dikaryotization.

The Massachusetts strain of avian infectious bronchitis virus was purified from embryonated hens′ eggs. Four major species of apparent mol. wt. 90000, 52000, 29000 and 26000 were resolved by SDS-polyacrylamide gel electrophoresis. Omission of reducing agent failed to resolve the 29000 mol. wt. component. Labelling of acrylamide gels with 125I-concanavalin A indicated that polypeptides of mol. wt. 90000, 29000 and 26000 were glycosylated and, in the absence of reducing agent, that the 29000 species migrated as a dimer in the 5000 mol. wt. region. Purified IBV radio-iodinated with Bolton and Hunter reagent, which banded as a single peak of radioactivity in Metrizamide gradients, was found to contain bands of radioactivity when analysed by SDS-PAGE, corresponding to the polypeptides of mol. wt. 90000, 52000 and 29000 resolved in stained gels. Disruption of IBV particles in Triton X-100 released two subviral particles, a 16 nm spike which comprised polypeptides of 90000, 52000 and 29000 mol. wt. and another denser spherical particle of 25 to 45 nm which contained RNA and the 52000 and 26000 polypeptides.