The collagen repeat sequence is a determinant of the degree of herpesvirus saimiri STP transforming activity.

Herpesvirus saimiri (HVS) is divided into three subgroups, A, B, and C, based on sequence divergence at the left end of genomic DNA in which the saimiri transforming protein (STP) resides. Subgroup A and C strains transform primary common marmoset lymphocytes to interleukin-2-independent growth, whereas subgroup B strains do not. To investigate the nononcogenic phenotype of the subgroup B viruses, STP genes from seven subgroup B virus isolates were cloned and sequenced. Consistent with the lack of oncogenic activity of HVS subgroup B viruses, STP-B was deficient for transforming activity in rodent fibroblast cells. Sequence comparison reveals that STP-B lacks the signal-transducing modules found in STP proteins of the other subgroups, collagen repeats and an authentic SH2 binding motif. Substitution mutations demonstrated that the lack of collagen repeats but not an SH2 binding motif contributed to the nontransforming phenotype of STP-B. Introduction of the collagen repeat sequence induced oligomerization of STP-B, resulting in activation of NF-kappaB activity and deregulation of cell growth control. These results demonstrate that the collagen repeat sequence is a determinant of the degree of HVS STP transforming activity.

Functional phenotype of transformed human alphabeta and gammadelta T cells determined by different subgroup C strains of herpesvirus Saimiri.

Based on sequence divergence in the transformation-relevant region, herpesvirus saimiri strains are classified into three subgroups. Only members of subgroup C transform human T lymphocytes to continuous interleukin-2-dependent growth in culture. In this study, human cord blood T cells were immortalized by using different subgroup C strains (C488, C484, and C139). The resulting T-cell lines represented different types of T-cell clones. They were either CD4+ or CD8+ and expressed either the alphabeta or the gammadelta type of T-cell receptors. If transformed by the same virus strain, alphabeta and gammadelta clones were similar with respect to viral persistence, virus gene expression, proliferation, and Th1-type cytokine production. However, major differences were observed in T cells immortalized by different subgroup C strains. Strain C139 persisted at low copy number, compared to the high copy number of prototype C488. The transformation-associated genes stpC and tip of strain C488 were strongly induced after T-cell stimulation. The homologous genes of strain C139 were only weakly expressed and not induced after activation. After CD2 ligation, the C488-transformed T cells produced interleukin-2, whereas the C139-transformed cells did not. Correspondingly, the C139-transformed T cells were less sensitive to cyclosporin A. Sequence comparison from different subgroup C strains revealed a variability of the stpC/tip promoter region and of the Lck-binding viral protein Tip. Thus, closely related subgroup C strains of herpesvirus saimiri cause major differences in the functional phenotype of growth-transformed human T cells.

Herpesvirus saimiri open reading frame 14, a protein encoded by T lymphotropic herpesvirus, binds to MHC class II molecules and stimulates T cell proliferation.

Herpesvirus saimiri (HVS) is an oncogenic, lymphotropic, gamma-herpesvirus that transforms human and simian T cells in vitro and causes lymphomas and leukemias in various species of New World primates. Nucleotide sequence analysis of the HVS genome revealed an open reading frame with 22% amino acid identity to the mouse mammary tumor virus 7 superantigen. In this study, we demonstrate that this open reading frame, HVS14, encodes a heavily glycosylated protein that is secreted. Both the HVS14 present in the supernatant of transfected cells and a chimeric HVS14.Fc fusion protein were found to bind to heterodimeric MHC class II HLA-DR molecules. The supernatant from HVS14-transfected cells induced the proliferation of human PBMC, which could be specifically inhibited by HVS14-specific mAbs. Purified peripheral blood T cells were induced to proliferate in the presence of accessory cells and HVS14-containing supernatant. Whereas the HVS14 protein stimulated T cell proliferation, the HVS14.Fc fusion protein blocked proliferative responses to soluble Ags in vitro. Collectively, these data indicate that HVS14 can function as an immunomodulator that may contribute to the immunopathology of HVS infection.

[Absence of herpesvirus saimiri STPC oncogene in salivary gland tumors and epithelial thymus tumors]. / Fehlen des Herpesvirus saimiri Onkogens STPC in Speicheldrüsen-tumoren und epithelialen Thymustumoren.

AIM AND METHODS: The oncoprotein STP-C-488 induces salivary gland and thymic epithelial tumours when expressed as a transgene in mice (MURPHY et al. 1994). Given the enigmatic tumorigenesis of corresponding tumours in humans, we now investigated genomic DNA and RNA from 11 thymomas, 5 pleomorphic adenomas and control autopsy material (n = 8) for the occurrence of the STP-C-488 sequences by Southern-blotting, Northern-blotting and PCR. RESULTS: All tumor samples and control tissues were negative for the STP-C-488 in Southern-blot and Northern-blot-hybridization. PCR analyses did not reveal amplification products of the length expected for STP-C-488. However, a PCR fragment of a different size was found in 50% of the thymomas and pleomorphic adenomas, but in only one of 8 controls. The sequence of this PCR product revealed local homologies with various herpesviruses. CONCLUSION: The oncoprotein STP-C-488 is not involved in the tumorigenesis of human thymomas and salivary gland tumours. Whether the novel sequences amplified preferentially from these tumours play a role in pathogenesis needs further investigation.

Alphaherpesvirus saimiri in rabbits: a model for human encephalitis?

One (KM91) of a series of isolates of alphaherpesvirus saimiri (alpha HVS) produced rapidly fatal encephalitis in rabbits following intradermal infection, whereas the others (KM180, KM322 and KM338) were non-lethal and produced ganglionitis and prolonged latency. Alphaherpesvirus saimiri KM91 initially produced ganglionitis but quickly ascended the spinal cord to the brain causing death 10 days post-infection. Prior infection with any of the three benign isolates or inoculation with beta-propiolactone (beta PL)-inactivated alpha HVS KM91 protected rabbits from lethal encephalitis when they were subsequently challenged with a lethal dose of alpha HVS KM91. Each of 20 rabbits co-inoculated in the same site with a lethal dose of alpha HVS KM91 and either alpha HVS KM322 (1.5 X 10(3) to 1.5 X 10(5) p.f.u.) or beta PL-inactivated alpha HVS KM322 (1 X 10(7) p.f.u. equivalents) survived. In contrast only half of those co-inoculated with alpha HVS KM91 and beta PL-inactivated alpha HVS KM91 (1 X 10(7) p.f.u. equivalents) survived. Co-inoculation of lethal alpha HVS KM91 (75 p.f.u.) and benign alpha HVS KM322 (1.5 X 10(3) p.f.u.) into opposite flanks resulted in protection from encephalitis in one of four rabbits. Alphaherpesvirus saimiri KM91 was shown to have the capacity to become latent in dorsal root ganglia if the rabbit did not die.

Detection of antibodies to Herpesvirus saimiri late antigens in human sera.

One hundred fifty sera from handlers of squirrel monkeys and 100 sera from individuals who had never handled monkeys were tested by immunofluorescence for antibodies reactive to structural proteins of Herpesvirus saimiri (HVS). Eleven (7.3%) of the occupationally exposed group and 4 (4%) of the noncontact group were seropositive for HVS by immunofluorescence assay, and 10 of these 15 (6.7 and 2%, respectively) were also seropositive for either the major glycoprotein (140 kD) or the major capsid protein (160 kD) of HVS by radioimmunoprecipitation assay. Two sera from handlers of squirrel monkeys, however, recognized many different HVS structural antigens by immunoprecipitation, and it seems unlikely that they could also be cross-reactive antibodies. Since these two sera did not contain antibodies to HVS early antigens or to the nonstructural antigens present in infected owl monkey kidney cells, and follow-up sera collected from the same individuals several months later were negative for antibodies to HVS, these individuals do not appear to have been infected by the virus. The risk that HVS poses to humans appears to be very low.

Characterization of four herpesviruses isolated from owl monkeys and their comparison with Herpesvirus saimiri type 1 (Herpesvirus tamarinus) and herpes simplex virus type 1.

Four herpesviruses were previously isolated from four outbreaks of lethal disease in owl monkeys. All four isolates have been shown to be antigenically closely related to each other and to Herpesvirus saimiri 1 (HVS-1) by kinetic neutralizations. The owl monkey strains also share similarities to HVS-1 and to each other with respect to host range, growth cycles and molecular weights of peptides and of DNA fragments generated by restriction endonuclease (R.E.) digestion. R.E. analysis, however, can differentiate strains by the use of certain enzymes. All four isolates share a common G-C ratio percentage with HVS-1 of 67 per cent. On the basis of these findings, we believe that these owl monkey virus isolates are strains of HVS-1.

Natural killer cells in relation to Herpesvirus saimiri-induced disease course and manifestations in owl monkeys.

Infection of owl monkeys with Herpesvirus saimiri (HVS) results in disease courses ranging from chronic infections to fatal cancers. Malignant disease manifestations include the appearance of transformed T-cells, suppressor T-cells, and virus-carrying cells in the peripheral circulation. The reasons for the variability in disease course remain unknown. This study examined natural killer (NK) functions in relation to disease manifestations following HVS infection. The results from lymphocyte fractionation studies indicated that the owl monkey NK cell was of T-cell lineage and had receptors for the Fc fragment of IgG. Following virus infection, NK activity was enhanced in parallel with the appearance of transformed and suppressor cells in monkeys that developed malignant disease. Further studies on the relationship of these three disease characteristics indicated that they involved at least partially different T-cell subpopulations. The results further indicated that the different disease manifestations induced by HVS were polyclonal and at least partially independent events.

Herpesvirus saimiri strain variability.

Herpesvirus saimiri was isolated from 22 squirrel monkeys by cocultivation of peripheral lymphocytes with permissive owl monkey kidney monolayer cells. Comparison of virion DNA fragments produced from restriction endonuclease digestion was used as a sensitive measure of strain variability. Although all isolates contained similarities and common features, 19 of the 22 were readily distinguished. Three of the isolates, however, were indistinguishable and possibly were related epidemiologically. Distinct subtypes of H. saimiri were not evident by these criteria; Peruvian, Colombian, Guyanan, and Bolivian squirrel monkeys yielded isolates without characteristic features peculiar to the geographic region. Three of three colony-born squirrel monkeys that were tested yielded a strain of virus distinct from that obtained from the mother. In separate experiments, two of three animals chosen at random yielded a strain of virus different from that originally obtained 16 and 22 months previously; only one of the three animals examined yielded the same strain of virus 22 months after the original isolation. The degree of restriction endonuclease fragment variability among H. saimiri strains appeared to be greater than previously observed for other herpesviruses.

Squirrel monkey retrovirus and Herpesvirus saimiri: observation in the same cell following isolation.

Electron microscopic examination of mink lung cells previously cultured with a squirrel monkey (Saimiri sciureus) throat swab suspension revealed the presence of squirrel monkey retrovirus (SMRV) and Herpesvirus saimiri (HVS) coexisting within the same cells in culture. HVS was identified by serum neutralization, and the retrovirus isolate was identified as SMRV by a morphologic examination, microimmunodiffusion analysis, and demonstration of an Mg2+ preference for the RNA-directed DNA polymerase.

Susceptibility of common marmosets (Callithrix jacchus) to oncogenic and attenuated strains of Herpesvirus saimiri.

Adult common marmosets, inoculated with either of 2 oncogenic Herpesvirus saimiri (HVS) strains, developed fatal lymphoproliferative disease within 23-25 days post inoculation (PI). The disease was identical to HVS-induced lymphoma in cotton-topped and white-lipped marmosets. Common marmosets inoculated with an attenuated HVS strain developed persistent infection; virus has been recovered from cocultivated lymphocytes of these animals for more than 384 days PI.

Herpesvirus saimiri-induced lymphoproliferative disease in howler monkeys.

Four of 5 howler monkeys (Alouatta caraya) experimentally infected with Herpesvirus saimiri (HVS) developed a rapidly fatal malignant lymphoma accompanied by peripheral T-cell lymphocytosis. HVS was isolated from fresh and tissue cultured blood and tissue lymphocytes and from cell cultures derived from nonlymphoid organs. Humoral antibodies against HVS-induced antigens were detected in the sera of the animals. The in vitro response of the peripheral blood lymphocytes to mitogenic stimulants was depressed following HVS infection.

The presence of Herpesvirus Saimiri genomes in virus-transformed cells.

Herpesvirus saimiri (H. salmiri) -transformed cells contained both types of viral DNA, unique L-DNA and highly repetetive H-DNA. DNA from spleen and lymph-node autopsies of two tumor-bearing marmoset monkeys contained 0.14-0.75% viral L-DNA -AND 0.115-1.08% H-DNA. This amount of H-DNA would be equivalent to the presence of 14-130 M-genomes per diploid tumor tissue cell. Six virus-transformed lymphoid cell lines, two of them virus-producing, contained 0.69-2.27% H-DNA and more than 0.72-1.95% L-DNA. These concentrations of H-DNA sequences correspond to 83-274 M-genome copies per lymphoid tissue culture cell. The majority of viral genomes in transformed non-producer lymphoid cell lines appeared to be defective, since part of the L-sequences present in virions were found to be deleted in the genome copies of transformed cells. There was a relative excess of repetitive H-sequences in all transformed cells in regard to the ratio of H-DNA/L-DNA in M-genomes of H. saimiri virions.

Mononuclear cell fraction carrying Herpesvirus saimiri in persistently infected squirrel monkeys.

Circulating lymphocytes from squirrel monkeys persistently infected with Herpesvirus saimiri (HVS) were separated into B- and T-lymphocyte fractions by a rosette-enrichment technique. HVS was isolated only from lymphocyte fractions forming rosettes or from unseparated lymphocytes; this indicated that T-lymphocytes were the target cells for HVS in the natural host, squirrel monkeys.

Absence of horizontal transmission of herpesvirus saimiri between experimentally infected and noninfected owl monkeys.

We did not detect cell-free Herpesviurs saimiri (HVS) in the oropharyngeal secretions of owl monkeys with leukemia or lymphoma induced by this virus. These animals failed to transmit either virus or disease to their uninoculated cage-mates or room-mates. Comparison of oropharyngeal secretions of HVS from owl monkeys and squirrel monkeys may provide insight as to how human herpesviruses are maintained in the oropharynx.

Herpesvirus saimiri-induced malignant lymphoma in rabbits.

Of 9 New Zealand White rabbits inoculated at multiple sc and im sites with a single dose of Herpesvirus saimiri (HVS), 2 developed malignant lymphomas 40-50 days post inoculation. At least 1 animal developed a terminal leukemic phase of the disease. HVS was isolated from the oral and conjunctival swabs and blood and tissue lymphocytes, but not from monolayer cell cultures derived from kidney or lung tissues of the diseased animals. The inoculated rabbits developed low titers of neutralizing antibodies against the virus. Antibodies against HVS specific early and late antigens were not detected in the sera of 7 animals that failed to develop clinical disease, but were detected in the serum of the 1 rabbit with lymphoma. The immunologic response of rabbits to HVS infection was compared to similar responses in infected nonhuman primates.

Experimental infection of squirrel and marmoset monkeys with attenuated Herpesvirus saimiri.

Herpesvirus saimiri (HVS) was propagated in vero cells for 3 passages at 39 degrees and cloned 3 times at 34 degrees. This virus was inoculated into cotton-topped marmoset and squirrel monkeys; all inoculated monkeys became infected as HVS was reisolated after their circulating lymphocytes were cultured with vero cells and measurable levels of antiviral antibodies developed that were measured by immunofluorescence and/or neutralization tests. None of the inoculated monkeys developed any signs of overt disease and all inoculated monkeys have survived 9 to 14 months postinoculation. The attenuated virus appears to be genetically stable as virus isolated from an infected marmoset was passed 3 times in vitro and then inoculated into other marmosets, which became infected and remained clinically well. Marmosets latently infected with attenuated HVS were not protected when challenged with a large dose (770 plaque-forming units) of oncogenic HVS, although these marmosets survived about 3 times longer than did inoculated control marmosets.

Response to primary infection with Herpesvirus saimiri in immunosuppressed juvenile and newborn squirrel monkeys.

Immunosuppression of juvenile squirrel monkeys with combined azathioprine, prednisolone, and antilymphocyte globulin resulted in decreased antibody responses to viral antigens after primary infection with Herpesvirus saimiri (HVS). The virus was repeatedly isolated from the oropharynx of immunosuppressed monkeys but not from untreated infected controls. Thus immune factors are important in inhibiting shedding of HVS from the oropharynx. HVS could be isolated from the peripheral blood lymphocytes of infected control monkeys but not from the lymphocytes of immunosuppressed monkeys. Immunosuppressed monkeys also had decreased percentages of lymphocytes capable of forming rosettes with sheep erythrocytes. These results indicate that the immunosuppressive agents had inhibitory effects on lymphocytes (presumably thymus derived) capable of being latently infected with HVS. Antibody responses in newborn monkeys infected with HVS were delayed compared with juvenile monkeys. Treatment of newborn monkeys with antilymphocyte globulin had no suppressive effect on antibody responses to HVS.