- Volume 45, Issue 1, 1979

The envelope spikes of Sindbis and Semliki Forest virus are arranged in a T=4 icosahedral surface lattice and, by deduction, it has been suggested that the nucleocapsid proteins are similarly arranged. After treatment of the virions with a non-ionic detergent the released nucleocapsids sediment in sucrose gradients at about 160S and 150S and have densities in CsCl of 1.42 g/ml and 1.425 g/ml, respectively, for Sindbis and Semliki Forest virus. At pH 6.0 Sindbis nucleocapsids do not contract like those of Semliki Forest virus. Nucleocapsids of both viruses are sensitive to the action of ribonuclease but only those of Semliki Forest virus undergo a drastic structural rearrangement due to the treatment. EDTA treatment in hypotonic conditions results in a decrease in the S-value for both particles. Electron micrographs show that the SFV nucleocapsids are partly ‘unfolded’ while those of Sindbis appear slightly contracted after exposure to EDTA.

The indirect immunofluorescence technique was used to investigate the expression of virus antigens in a Swiss albino mouse strain infected by mouse mammary tumour virus (MMTV). The antisera used were monospecific for: the glycopolypeptides gp160 and gp47, and the internal polypeptides p28 and p8.

In mice infected with milk-borne MMTV, all four antigens were detected in the mammary gland during the first pregnancy and then throughout life, and in mammary tumours. Antigen gp160, located at the secretory border of all glandular cells, is shared by the plasma membrane of the mammary cell, whether virus-producing or not, and by the virus envelope, as shown by the use of absorbed antisera. The other three antigens are virus-specific. Anti-p47 serum stained the secretory border of cells whereas the fluorescence related to p28 and p8 consisted of a few large intracytoplasmic spots. The specific antibodies for p28 and p8 were only absorbed by disrupted virions, confirming that these polypeptides are internal antigens.

With serial sections of glands from pregnant and old female mice, the alveoli stained with anti-p28 or anti-p8 serum were found to react also with anti-gp47 serum. However, during lactation, the presence of p28 and p8 could not be detected in the alveoli stained with anti-gp47 serum. Thus, infected alveoli express virus antigens differently. In the mammary glands of all mice studied, some alveoli did not display any staining when the latter three antisera were used. The specific antigenic pattern, maintained in differentiated mammary tumours, became a diffuse and unlocalized staining after transformation into the undifferentiated state.

With the exception of the kidney, where gp47 was found in the glomeruli, all other organs of the mouse did not stain with any of the antisera. The presence of gp47 in the glomeruli, probably related to immune-complex deposits, increases with the age of the mouse and is most noticeable in tumour-bearing females

In mice free of milk-borne MMTV, examined during the first week of lactation, antigen gp47 could not be detected. Internal antigens p28 and p8 were detected in nearly all mammary cells as numerous small cytoplasmic dots. This suggests a limited expression of an endogenous virus genome; this expression would be modified when milk-borne virus is produced.

Extracts of various organs, mammary tumours and sera from milk-borne MMTV infected Swiss albino mice of different age, sex and physiological conditions were tested by radioimmunoassay for the presence of gp47, the main envelope polypeptide, and p28, the main core protein of the virus. Except in brain, ovaries and testes, both antigens were found in all organs of old animals and of females after the onset of their first pregnancy. Antigens were not present in organs of weanlings or in whole foetuses. Higher values were found in mammary glands, mammary tumours, epididymis and seminal vesicles. These organs also harboured a greater amount of gp47 than p28. The serum generally contained gp47 but rarely p28. This indicates that gp47 is not virion-bound in blood. Pregnancy, lactation and especially the presence of mammary tumours increased the concentration of gp47 in serum. The results do not allow localization of target organs of MMTV infection in the interval between ingestion of the virus by the suckling mouse and the first pregnancy. Moreover, results obtained with one group of mice devoid of exogenous virus show that, as endogenous MMTV genome expresses p28, it might account for part of the p28 detected exogenous MMTV-infected mice.

The DNA of red cowpox virus strain Brighton or its white pock variant was analysed by cleavage with restriction endonucleases HindIII, Xho1, Pst1 or Kpn1. Physical maps were constructed and the genomes compared with that of vaccinia virus strain DIE. The size of the red cowpox genome is 23 to 29 megadaltons greater than that of vaccinia and results from the presence of additional, near terminal sequences. An internal region of about 75 megadaltons appears to be highly conserved between the two viruses. The red cowpox genome contains near terminal, repetitive sequences which have some homology with those of vaccinia virus DNA. Rapid renaturation of red cowpox terminal restriction fragments indicates that these are covalently cross-linked.

Viable white pock variants arise continually and map as deletion mutants lacking similar sequences from one specific terminus only of the parental genome. The deletion represents 11 to 12% of the red cowpox DNA and includes the terminal repetition which therefore is not required for replication. The deleted terminus of the white pock variant genome does not appear to be cross-linked.

Complement-dependent antibody lysis of vaccinia-infected cells was examined to demonstrate the antigenic differences between extracellular (ECV) and intracellular (ICV) forms of vaccinia virus. Cytolytic antibodies present in the antisera raised against ECV or live virus (LV) were completely removed by absorption with infected cell membranes or purified ECV but not with purified ICV. Absorption with infected cell membranes also abolished the neutralizing activity of ECV and LV antisera against ECV. On the other hand, antiserum against ICV did not contain cytolytic antibodies against vaccinia-infected cells, even though its neutralizing antibody titre against ICV was high. Moreover, both ECV and ICV anti-sera neutralized a small proportion of the heterologous form of virus, despite using purified preparations of ECV and ICV, respectively, for raising these antisera in rabbits. In contrast, the 51Cr release test only detected the antibodies against ECV and thus can be used to differentiate between the antibody activity of a serum against ECV and ICV.

Virus-immune sera applied to infected target cells inhibited Tc cell-mediated lysis in vitro. Blocking activity was clearly present in serum by 6 days after ectromelia virus infection. Activity was found in the IgG and IgM fractions of hyperimmune sera and was specific for the immunizing virus when ectromelia and influenza viruses were used, but did not distinguish between the serologically related poxviruses ectromelia, vaccinia and rabbitpox. There was no requirement for the donors of immune serum to be of the same mouse strain as the target cells, or the same species, since rabbit sera blocked similarly to mouse sera.

These findings imply that virus-specified antigens recognized by B cells are physically close to, or identical to, the virus antigens involved in Tc cell-recognizable antigenic changes in infected cell surfaces. There was no evidence for modified H-2 molecules alone, or other proteins coded by derepressed host cell genes being recognized by virus-immune Tc cells. Significant inhibition of lysis by anti-viral antibody was only observed on fibroblast type target cells. Macrophage and P815 targets were refractory to blocking. These findings are discussed in practical terms and in relation to possible regulation of Tc cell responses in vivo by antiviral antibody.

Human osteogenic sarcoma cells transformed by murine sarcoma virus (R970) showed restricted growth of human cytomegalovirus (HCMV). Virus was attached to the same extent as in human fibroblasts. HCMV growth was blocked at early stages after virus penetration. Splitting of infected R970 cultures resulted in infection of all cells. In experiments using synchronized R970 cells it was found that factor(s) associated with the S-phase of the cell cycle might be necessary for establishment of the infection.

After immunization with Sindbis virus, T-cell deficient nude mice, compared to normal littermates, were equally protected against challenge with Sindbis virus. However, the nude mice showed about one-tenth the protection observed with normal littermates after challenge with Semliki Forest virus at a dose of 100 LD50. This is consistent with our previous interpretation that sensitized T-cell populations play a major role in cross protection between the two togaviruses. The remaining low level of specific cross protection in nude mice (detectable only at a challenge dose of 10 LD50) could not be attributed to an anamnestic response of neutralizing antibody to the challenge virus or to an effective antibody-dependent, complement-mediated cytolysis of infected cells in vivo. Other possible compensatory mechanisms to explain the low level of specific cross protection in nude mice are discussed.

When an avian sarcoma virus (ASV), subgroup C Bratislava 77 (B77-C) was inoculated into duck embryo fibroblast cultures (DEF) at a m.o.i. of 0.02, its replication was retarded by about 3 days compared with that in chick embryo fibroblast cultures (CEF). A transformation-defective (td) mutant was isolated during this period of retardation. Unlike the sarcoma virus, this td mutant replicated in both DEF and CEF with no retardation, even at a low m.o.i. The subgroup C Prague strain of Rous sarcoma virus (PR-C), which can infect DEF, also replicated in DEF slower than its td mutant, tdPR-C, at a m.o.i. of 0.02.

Poliovirus type 1, vaccine strain (LSc, 2ab), which is a temperature- and actinomycin D-sensitive mutant derived from type 1 Mahoney strain, was grown in HeLa cells in the presence of 32P and a low concentration of actinomycin D. Seven and a half h p.i., genome 32P-RNA was recovered from the purified virion. Analysis of RNase T1 digests of the RNA by two-dimensional gel electrophoresis revealed that three possible point mutation sites exist in the large and unique oligonucleotides in the fingerprint. Neither a capping structure nor a nucleotide such as pppNp, ppNp or pNp, was detected by ion exchange column chromatography at pH 5.0 after digestion of virion RNA with RNase T2. Instead, a 32P-labelled compound, which could be digested with Pronase or proteinase K, was eluted at the void volume of the column. Proteinase K digests of the 32P-labelled compound contained pUp or pU as a single labelled compound, when genome RNA was digested with RNase T2 or nuclease P1, respectively, before digestion with the proteinase.

Our data locate possible point mutation sites on the genome of a mutant strain (LSc, 2ab) of type 1 poliovirus and show that a protein (VPg) is covalently bound to the 5′-terminus of the RNA. The protein (VPg) of LSc, 2ab strain migrates faster than capsid protein VP4 (mol. wt. 7000 to 8000) in a polyacrylamide gel and is thus similar to the VPg of the wild-type virus.

DNA of Marek’s disease virus (MDV) was compared to that of herpes virus of turkey (HVT). Centrifugation of the two virus DNAs in neutral glycerol and CsCl density gradients showed that the MDV genome was slightly larger than that of HVT and that the buoyant density (1.705 g/ml) of MDV DNA in CsCl gradients was slightly lower than that (1.707 g/ml) of HVT DNA. MDV and HVT DNAs were digested with either EcoRI or HindIII restriction endonuclease and analysed by 0.5% agarose gel electrophoresis. The cleavage patterns of HindIII or EcoRI DNA digests of two strains of these two viruses showed general similarities between the strains, but not between MDV and HVT. However, a few fragments of EcoRI or HindIII digests of MDV DNA co-migrated with those of HVT DNA. DNA-DNA reassociation kinetics and DNA-RNA hybridization between these two viruses indicated that MDV and HVT DNAs share detectable homology, although it is less than 5%. The DNA of a HVT variant, which has lost the ability to protect chickens from Marek’s disease, appeared similar to DNA of the vaccine strain in size and buoyant density and in its restriction endonuclease cleavage pattern.

The progeny from mixed infections of tobacco protoplasts with a temperature sensitive (ts) and a temperature resistant (wt) strain of cowpea chlorotic mottle virus at restrictive temperatures (35 °C) was largely (90%) ts, judged by the coat protein produced by single lesion isolates grown at 25 °C. However, although apparently ts at 25 °C, many isolates were temperature resistant at 35 °C and produced virus. Successive ‘purification’ through single lesions allowed normal ts or wt viruses to be recovered from these unusual isolates. The genomes of ts and wt appear able to co-exist in mixed infections; ts is dominant over wt at all temperatures so that most of the progeny has ts genome, but wt remains available to rescue ts under restrictive conditions. There was no evidence that the phenomenon was due to recombination.

Mammalian cells transformed with either Prague strain Rous sarcoma virus of subgroup C (XC cells) or Schmidt-Ruppin strain Rous sarcoma virus of subgroup D (RSCH cells) yielded virus upon fusion with chick cells. Virus was also rescued by transfection of DNA from these cells on to chick cells. However, virus rescue did not occur upon transfection of duck cells, and fusion with duck cells led to virus rescue only from RSCH and not from XC cells. To investigate this restriction on duck cells the non-defective Prague strain of Rous sarcoma virus of subgroup C (PR-RSV-C) was adapted for efficient replication in duck embryo cells (daPR-RSV-C) by long-term passage in vitro. However, a second PR-RSV-C isolate, rescued from the rat XC sarcoma line (XC DNA 940 virus), failed to adapt to growth in duck cells. When transformed with daPR-RSV-C, which replicates in duck cells as well as in brown leghorn embryo (BLEF) cells, duck cells yielded DNA which transfects fresh duck cells, in contrast to DNA isolated from chicken or duck cells transformed with parental PR-RSV-C.

The synthesis of 3H-uridine-labelled complete virus, virus RNA and presumed replicative form (RF) were studied during the replication of southern bean mosaic virus (SBMV) in soybean callus cells. The inoculated cells were pre-incubated at 6 °C for 4 days and then moved to 25 °C to improve the synchronization of virus multiplication. The synthesis of complete virus, measured by the incorporation of 3H-uridine into virions and by infectivity assays, rose rapidly during 0 to 34 h after transfer to 25 °C. An RNA with a mol. wt. approximating to that of the postulated RF of SBMV-RNA and exhibiting partial resistance to RNase digestion was synthesized in significant amounts. This occurred after a 16 to 24 h incubation, preceding the major period of virus RNA synthesis which reached maximum during 40 to 48 h. Pulse-chase experiments, within limits, suggested the possible precursor role of the postulated RF in the synthesis of virus RNA. Accumulation of an RNA with electrophoretic properties similar to the presumptive RF of SBMV-RNA, was found in inoculated cells incubated at 6 °C from 84 to 96 h, suggesting a possible blockage of virus replication at the double-stranded RNA stage in these cells.

Evidence is presented supporting the hypothesis that reovirus intermediate subviral particles (ISVP), which show increased infectivity relative to intact virions, can gain entry into host L cells by two alternative pathways. One pathway is by the process of viropexis, involving phagocytic vacuoles. A second entry pathway is via direct penetration of the plasma membrane of the cell, without involvement of a phagocytic vacuole. Using electron microscopy, a kinetic analysis of the uptake process was carried out. Results indicate that at 37 °C ISVP gain entry into host cells primarily by direct entry, although viropexis also occurs, while intact virions gain entry by viropexis almost exclusively. A second line of experimental evidence consistent with the idea that ISVP can ‘melt’ their way through the plasma membrane is provided by studies on the release of pre-loaded radioactive 51Cr from host cells following infection. 51Cr release data demonstrate that infection with ISVP leads to an immediate increased leakiness of the cell plasma membrane, whereas no such increase takes place following infection with an equivalent number of intact virions. This demonstrates that ISVP can interact with the plasma membrane of the cell in a manner which is qualitatively different from the interaction between intact virions and the plasma membrane.

The ability of ISVP to directly penetrate the plasma membrane of the host cell, which intact virions apparently cannot do, could explain the decreased duration of the eclipse phase, as well as the increased infectivity of ISVP, relative to that observed for infection with intact virions.

Human leukocyte interferon attaches to hyperimmune serum immunoglobulins, coupled to agarose, even though the antibodies are not specific for interferon. Unless it is first washed off with ethylene glycol, the attached interferon elutes together with immune-specific antigens during immuno-affinity chromatography.

5-Bromodeoxyuridine (BrdUrd) increased interferon production by the Namalwa line of human lymphoblastoid cells treated with Sendai virus, but inhibited their growth. Thymidine, which also inhibited cell growth had no effect on interferon production, so that growth inhibition per se was not the cause of the stimulation. BrdUrd was incorporated into cellular DNA; 5-chlorodeoxyuridine and 5-iododeoxyuridine (which are also incorporated) increased the interferon yield, but 5-fluorodeoxyuridine (which is not incorporated) did not. Thymidine reduced both the incorporation of BrdUrd and its stimulatory effect on interferon production. Deoxycytidine (which prevents the cytotoxic effects of BrdUrd) had no effect on the stimulation. BrdUrd also stimulated interfer on production in response to poly(rI).poly(rC) in growing human diploid fibroblasts but not in SV40 virus-transformed human cells. Since BrdUrd was incorporated into the DNA of all these cells, we concluded that incorporation is necessary, but not sufficient for the stimulation of interferon formation.

A non-virogenic African green monkey kidney cell line BGM/MV persistently infected with a neurotropic mouse brain-adapted strain of measles virus, was found to have undergone significant changes in the virus-host cell relationship between passages 35 and 119. Rather than the stable non-cytopathic relationship previously reported in which approximately 100% of the cells contained measles antigens and < 1% of the cells expressed cell surface measles antigen, we observed cyclic manifestations of c.p.e. together with changes in the percentage of cells expressing intracellular and cell surface measles antigens. Treatment of BGM/MV cells with actinomycin D effected an increase in the percentage of cells expressing cell surface virus haemagglutinin (HA) at times when the percentage of cells with surface HA was less than the percentage of cells with intracellular measles antigens. Super-infection studies employing measles virus and vesicular stomatitis virus revealed a consonant cyclic refractivity and essentially no refractivity, respectively. Endogenous, infectious measles virus was not detected nor was interferon. It was concluded that a host cell factor other than interferon was modulating the cyclic expression of the measles virus infection.