The latency-associated nuclear antigen homolog of herpesvirus saimiri inhibits lytic virus replication.

Herpesvirus saimiri (HVS), a T-lymphotropic tumor virus of neotropical primates, and the Kaposi's sarcoma-associated human herpesvirus 8 (KSHV) belong to the gamma-(2)-herpesvirus (Rhadinovirus) subfamily and share numerous features of genome structure and organization. The KSHV latency-associated nuclear antigen (LANA) protein appears to be relevant for viral persistence, latency, and transformation. It binds to DNA, colocalizes with viral episomal DNA, and presumably mediates efficient persistence of viral genomes. LANA further represses the transcriptional and proapoptotic activities of the p53 tumor suppressor protein. Here we report on the ORF73 gene of HVS strain C488, which is the positional and structural homolog of KSHV LANA. The ORF73 gene in OMK cells can encode a 62-kDa protein that localizes to the nucleus in a pattern similar to that of LANA. We show that the ORF73 gene product can regulate viral gene expression by acting as a transcriptional modulator of latent and lytic viral promoters. To define the HVS ORF73 function in the background of a replication-competent virus, we constructed a viral mutant that expresses ORF73 under the transcriptional control of a mifepristone (RU-486)-inducible promoter. The HVS ORF73 gene product efficiently suppresses lytic viral replication in permissive cells, indicating that it defines a critical control point between viral persistence and lytic replication.

Persistence of selectable herpesvirus saimiri in various human haematopoietic and epithelial cell lines.

Herpesvirus (h.) saimiri, an infectious agent of squirrel monkeys, is capable of persisting in T lymphocytes of various primate species. It has been used as a vector for the functional analysis of regulatory genes in primary human T lymphocytes. As it is not yet known whether other cell types are capable of supporting viral persistence, various human cell lines were investigated using selectable h. saimiri recombinants. The lines chosen represent cells from the epithelium and connective tissue as well as from all haematopoietic lineages, i.e. cells of B and T lymphoid origin as well as myeloid-, fibroblast- and carcinoma-derived cultures converted to Geneticin or hygromycin B resistance, and harbouring episomal DNA of the selectable recombinants. The Burkitt's lymphoma-derived cell line Raji also contained simultaneously persisting episomes of the Epstein-Barr virus. Most of the cell cultures except a pancreatic carcinoma line and foreskin fibroblasts did not produce infectious virus. These observations show that a herpesvirus genome can persist episomally in a broad range of cultured cell types. The variety of infectable cell types and species suggests the presence of a widely distributed and well conserved virus receptor for h. saimiri. Thus the h. saimiri genome could be applied more generally as a vector.

The effects of 5-azacytidine, 12-O-tetradecanoylphorbol 13-acetate and sodium n-butyrate on reactivation of alphaherpesvirus saimiri from explant cultures of latently infected rabbit dorsal root ganglia.

The DNA hypomethylating agent 5-azacytidine greatly increased the reactivation of alphaherpesvirus saimiri-1 (alpha HVS) from latently infected rabbit dorsal root ganglia, although it inhibited the virus yield and plaque formation efficiency of alpha HVS in Vero cells. 12-O-Tetradecanoylphorbol 13-acetate (a protein kinase C activator) and sodium n-butyrate both had a stimulating action on replication in Vero cells but did not affect the release of alpha HVS from latently infected rabbit dorsal root ganglia.

Asynchronous expression of the immediate-early protein of herpesvirus saimiri in populations of productively infected cells.

The time course of virus replication in cultures of permissive cells infected with high multiplicities of herpesvirus saimiri (HVS), a gammaherpesvirus, is protracted relative to the replication of herpes simplex virus (HSV), an alphaherpesvirus, under similar conditions. The basis for this difference was investigated by quantitative immunofluorescence microscopy exploiting monoclonal antibodies specific to the HVS 52 000 mol. wt. immediate-early polypeptide (IE 52K) and to delayed-early (DE 51K, DE 110K) and late (130K and capsid proteins) gene products to measure the timing of gene expression in individual cells of infected cultures. The timing of the transitions from IE to DE and from DE to late protein synthesis occurred at proportionately different intervals in the growth cycle of HVS, relative to that of HSV. In particular, the DE to late transition occurred relatively later in HVS infections. However, asynchrony in the events leading to the expression of the first class of virus proteins (IE 52K) was the main source of the extended course of HVS replication in populations of infected cells. This asynchrony was not modified significantly by infection at different stages of the host cell-cycle and was reduced, but not overcome, by very high applied multiplicities of infection. Double-antibody staining revealed a positive correlation between the accumulation of high concentrations of parental virus particles at perinuclear sites and early detection of HVS IE 52K gene expression. Both of these events remained sensitive to a microtubule poison (colcemid) for many hours after infection with HVS, whereas the rapid and synchronous expression of the IE 175K protein (ICP4) in HSV-1-infected cells was insensitive to post-infection exposures to this drug. We conclude that significant differences in early stages of virus entry and intracellular processing which precede immediate-early gene expression are largely responsible for differences between the replicative cycles of these representatives of gamma- and alphaherpesviruses in cultures of permissive cells.

Interaction of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) with owl monkey kidney cells in enhancing the yields of Herpesvirus saimiri (HVS) and its antigens.

Pre- and posttreatment with N-methyl-N'-nitro-nitrosoguanidine (MNNG) of owl monkey kidney (OMK) cells infected with Herpesvirus saimiri (HVS) resulted in one to three logs higher yields of virus, depending upon the dose of MNNG. A higher percentage of cells also showed HVS early antigen (EA) and late antigen (LA) by immunofluorescence when OMK cells infected with HVS were fed with medium containing MNNG. The high yields of HVS were also observed by electron microscopy. MNNG did not induce HVS-EA in HVS nonproducer lymphoblastoid T cells, nor did it enhance TPA-induced EA to LA. The data suggest that MNNG could be useful in obtaining high yields of virus and/or antigen-producing cells for immunofluorescence or other biochemical experiments, especially from those strains of HVS which grow poorly in vitro. The interaction of MNNG and HVS could also be useful for in vitro transformation or in vivo enhancement of the malignant process.

The antiviral spectrum of (E)-5-(2-bromovinyl)-2'-deoxyuridine.

The antiviral activity spectrum of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) is not restricted to herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) but also encompasses several other herpesviruses such as suid herpesvirus type 1 (SHV-1), bovid herpesvirus type 1 (BHV-1), simian varicella virus (SVV), herpesvirus saimiri, herpesvirus platyrrhinae, and the baculovirus Trichoplusia ni multiple nuclear polyhedrosis virus. Other herpesviruses such as herpes simplex virus type 2, cytomegalovirus and Epstein-Barr virus are also inhibited by BVdU, albeit to a lesser extent than HSV-1, VZV, SHV-1, BHV-1 and SVV.

Epstein-Barr virus and Herpesvirus saimiri: sensitivity to interferons and interferon inducers.

The in vitro sensitivity of oncogenic herpesviruses, Epstein-Barr virus (EBV), and Herpesvirus saimiri (HVS) to human interferon produced by normal human leukocytes (Le), lymphoblastoid cell lines (LYI), and diploid fibroblasts (Fi) was studied. Four virus strains were used: HVS S295C, the highly oncogenic HVS S396-O, the transforming B95-8 strain of EBV, and the nontransforming P3HR1 strain of EBV. All interferons were active when applied to the cells after absorption of HVS and P3HR1-EBV, although different amounts were required to achieve 50% inhibition of HVS-induced cytopathic effect or EBV-induced early antigen (EA) expression. Transformation of human umbilical cord blood lymphocytes (HCBL) by the B95-8 strain of EBV was prevented only by Le and LYI. In these experiments, the most effective inhibitor of the oncogenic herpesviruses was Le, and the least effective was Fi. The effect of polynucleotides poly(I).poly(C) and the complex of poly(I).poly(C) with poly-L-lysine and carboxymethylcellulose on HVS and EBV was also studied. Their inhibitory action was proportionate to the ability of herpesvirus-infected cells to produce interferon. Thus owl monkey kidney cells, which produce relatively high levels of interferon, required nanogram quantities of polynucleotides to become resistant to HVS. Transformation of HCBL by B95-8-EBV was also prevented by poly(I).poly(C). In Raji cells superinfected with P3HR1-EBV, polynucleotides failed to stimulate interferon, and higher EBV-induced EA expression was observed. The percentage of P3HR1 and Raji cells spontaneously expressing EBV-associated antigens remained unchanged after exposure to either interferon or polynucleotides.

Comparative studies of interferon and three antiviral agents on neurotropic and oncogenic herpesviruses.

The antiviral effects of four compounds, phosphonoacetate, phosphonoformate, acycloguanosine, and purified human lymphoblastic interferon, were tested against two neurotropic herpesviruses, herpesvirus platyrrhine and herpes simplex virus, and two oncogenic herpesviruses, herpesvirus saimiri and herpesvirus ateles. All four compounds induced different degrees of inhibition of these herpesviruses. Phosphonoacetate and phosphonoformate at concentrations of 50 micrograms/ml or greater showed powerful antiviral activities. Interferon was more effective against herpesvirus saimiri and herpesvirus ateles, the two oncogenic viruses. Herpes simplex virus and the oncogenic herpesviruses were effectively inhibited by acycloguanosine, whereas herpesvirus platyrrhine proved to be resistant. The simian herpesviruses required a higher concentration of phosphonoformate, phosphonoacetate, and acycloguanosine for antiviral action. The antiviral action of all four compounds was contingent on the continued presence of the compounds in the infected cell culture medium.

Inhibition of Herpesvirus saimiri replication by phosphonoacetic acid, benzo(a)pyrene, and methylcholanthrene.

The present investigations were undertaken to determine the possible effects of two carcinogenic polycyclic aromatic hydrocarbons, benzo(a)pyrene and 3-methylcholanthrene on Herpesvirus saimiri replication. The results from these experiments were compared with the effects of phosphonoacetic acid on the virus replication cycle. Phosphonoacetic acid inhibited the synthesis of virus-induced intracellular late antigens, membrane antigens and infectious virus but not the synthesis of the early antigens induced by H. saimiri. In contrast, benzo(a)pyrene and 3-methylcholanthrene inhibited primarily membrane antigen expression and infectious virus production. Benzo(a)pyrene was the most effective of the two compounds, with significant inhibition occurring with 2 mug/ml, whereas a minimum concentration of 10 mug/ml was required with 3-methylcholanthrene. Both compounds were most effective when present continuously during the 4-day infection process. However, exposure of infected cultures to a 3-hr pulse with each chemical also inhibited membrane antigen expression. Furthermore, pretreatment of cells for 48 hr before virus infection resulted in the inhibition of membrane antigen expression but not that of early or late antigens. These result demonstrate that some carcinogenic chemicals are capable of altering the H. saimiri replication cycle, primarily by inhibiting some but not all late events.

Sequential immunofluorescence and infectivity studies on the replication of Herpesvirus saimiri in owl monkey kidney cells.

Methods are described for the preparation and authentication of a highly specific antiserum against herpesvirus saimiri (HVS) capsid antigens. The antiserum was used in immunofluorescence tests to follow the development of capsid antigens in HVS-infected owl monkey kidney cells throughout the virus replication cycle in parallel with sequential titrations of virus infectivity in both cells and medium. Fluorescence was detected as a round or oval, bright green area of staining at the centre of the nucleus which was similar in outline to the Cowdry type A inclusion seen in HVS-infected cells stained by haematoxylin and eosin. The first detection of fluorescence towards the end of the eclipse phase of the virus growth cycle, and its abolition by the treatment of infected cultures with cytosine arabinoside confirmed the identity of HVS capsid antigens as late antigens. The failure to detect fluorescence in the cytoplasm of HVS-infected cells has brought to light a conflict between the site of accumulation of virus capsid antigens as determined by immunofluorescence and the finding, by electron microscopy, of cytoplasmic immature particles in intact cells during the early stages of the virus replication cycle. The significance of this discrepancy is discussed in relation to its possible existence for other members of the herpesvirus group.

Preventive vaccination against Herpesvirus saimiri-induced neoplasia.

In this paper we discuss the use of Herpesvirus saimiri as a model for the development of vaccines against herpesvirus-induced neoplasia in primates. Attempts at protection against the oncogenicity of H. saimiri have centered on the inactivation of virus by heat and formalin, the production of temperature-sensitive H. saimiri mutants, and the attenuation of the virus. Each of these approaches has provided information of use in the development of vaccines that may possibly be used in man.

Nonhuman primate models for lymphoma, leukemia, and other neoplasms.

Herpesvirus saimiri (HVS) is an oncogenic virus for a variety of nonhuman primates. HVS does not produce overt disease upon inoculation in the natural host (squirrel monkey) but consistently induces neoplasms including lymphomas and lymphocytic leukemias in 4 other species of monkeys. Various drugs inhibit replication of HVS in vitro including cytosine arabinoside and adenine arabinoside. In addition, the lymphoma and leukemia induced in owl monkeys responds to vincristine and prednisolone, cyclophosphamide, cytosine arabinoside, and human interferon. Of the various chemical carcinogens studied, the antitumor agent procarbazine induces neoplasms in a variety of species including monkeys. Thus far this compound has induced acute myelogenous leukemia (AML), lymphoma, and hemangiosarcomas in macaques. We have induced primary liver tumors in macaques with several nitrosamines and aflatoxin B1 and these tumors produce alpha-fetoprotein (AFP) which can be assayed for both diagnosis and therapy. Thus far, therapy of hepatocellular carcinoma has been most successful with surgical resection; and the tumor mass and serum AFP have been less responsive to single agent chemotherapy. These nonhuman primate models are useful for an understanding of the cause, diagnosis, prevention, and treatment of the human disease.