Owl Monkey Kidney Cells Research Articles

Characterization of HIV-1 uncoating in human microglial cell lines

BackgroundAfter viral fusion with the cell membrane, the conical capsid of HIV-1 disassembles by a process called uncoating. Previously we have utilized the CsA washout assay, in which TRIM-CypA mediated restriction of viral replication is used to detect the state of the viral capsid, to study the kinetics of HIV-1 uncoating in owl monkey kidney (OMK) and HeLa cells. Here we have extended this analysis to the human microglial cell lines CHME3 and C20 to characterize uncoating in a cell type that is a natural target of HIV infection.MethodsThe CsA washout was used to characterize uncoating of wildtype and capsid mutant viruses in CHME3 and C20 cells. Viral fusion assays and nevirapine addition assays were performed to relate the kinetics of viral fusion and reverse transcription to uncoating.ResultsWe found that uncoating initiated within the first hour after viral fusion and was facilitated by reverse transcription in CHME3 and C20 cells. The capsid mutation A92E did not significantly alter uncoating kinetics. Viruses with capsid mutations N74D and E45A decreased the rate of uncoating in CHME3 cells, but did not alter reverse transcription. Interestingly, the second site suppressor capsid mutation R132T was able to rescue the uncoating kinetics of the E45A mutation, despite having a hyperstable capsid.ConclusionsThese results are most similar to previously observed characteristics of uncoating in HeLa cells and support the model in which uncoating is initiated by early steps of reverse transcription in the cytoplasm. A comparison of the uncoating kinetics of CA mutant viruses in OMK and CHME3 cells reveals the importance of cellular factors in the process of uncoating. The E45A/R132T mutant virus specifically suggests that disrupted interactions with cellular factors, rather than capsid stability, is responsible for the delayed uncoating kinetics seen in E45A mutant virus. Future studies aimed at identifying these factors will be important for understanding the process of uncoating and the development of interventions to disrupt this process.

Tanapoxvirus lacking a neuregulin-like gene regresses human melanoma tumors in nude mice.

Neuregulin (NRG), an epidermal growth factor is known to promote the growth of various cell types, including human melanoma cells through ErbB family of tyrosine kinases receptors. Tanapoxvirus (TPV)-encoded protein TPV-15L, a functional mimic of NRG, also acts through ErbB receptors. Here, we show that the TPV-15L protein promotes melanoma proliferation. TPV recombinant generated by deleting the 15L gene (TPVΔ15L) showed replication ability similar to that of wild-type TPV (wtTPV) in owl monkey kidney cells, human lung fibroblast (WI-38) cells, and human melanoma (SK-MEL-3) cells. However, a TPV recombinant with both 15L and the thymidine kinase (TK) gene 66R ablated (TPVΔ15LΔ66R) replicated less efficiently compared to TPVΔ15L and the parental virus. TPVΔ15L exhibited more robust tumor regression in the melanoma-bearing nude mice compared to other TPV recombinants. Our results indicate that deletion of TPV-15L gene product which facilitates the growth of human melanoma cells can be an effective strategy to enhance the oncolytic potential of TPV for the treatment of melanoma.

Complementary assays reveal a relationship between HIV-1 uncoating and reverse transcription

During the early stages of HIV-1 replication the conical capsid composed of p24(CA) protein dissociates from the rest of the cytoplasmic viral complex by a process called uncoating. Although proper uncoating is known to be required for HIV-1 infection, many questions remain about the timing and factors involved in the process. Here we have used two complementary assays to study the process of uncoating in HIV-1-infected cells, specifically looking at the timing of uncoating and its relationship to reverse transcription. We developed a fluorescent microscopy-based uncoating assay that detects the association of p24(CA) with HIV-1 viral complexes in cells. We also used an owl monkey kidney (OMK) cell assay that is based on timed TRIM-CypA-mediated restriction of HIV-1 replication. Results from both assays indicate that uncoating is initiated within 1 h of viral fusion. In addition, treatment with the reverse transcriptase inhibitor nevirapine delayed uncoating in both assays. Analysis of reverse transcription products in OMK cells revealed that the generation of early reverse transcription products coincides with the timing of uncoating in these assays. Collectively, these results suggest that some aspect of reverse transcription has the ability to influence the kinetics of uncoating.

Differential Susceptibility of Human Cancer Cell Lines to Wild-Type Tanapoxvirus Infection~!2009-09-05~!2010-02-23~!2010-04-08~!

Tanapoxvirus (TPV) is a member of the genus Yatapoxvirus in the family Poxviridae and is endemic to equatorial Africa. This disease is restricted to human and non-human primates, producing a mild febrile illness characterized by a single or more rarely additional pock-like lesions on the extremities. While there are several studies elucidating the replication cycle and host range of TPV, there is currently no standardized investigation comparing the ability of TPV to successfully replicate in a variety of tumor cell lines. This study examined the cytopathic effect and calculated the efficiency of TPV replication in vitro using 14 different human cancer cell lines. TPV replicates efficiently in some human tumor cells, and is restricted in others when measured by viral titer at 7 days post infection. Results described here clearly demonstrate that TPV replication in one glioblastoma cell line (U-373), and one colorectal cancer cell line (HCT-116) is more productive than in owl monkey kidney cells (OMK). Replication in two renal cancer cell lines (ACHN and Caki-1) is also increased when compared to OMK. TPV infection produced the greatest change in cellular morphology in U-373 cells, and to a much lesser degree in the breast cancer cell lines T-47D and MCF-7, and in the ovarian cancer line SK-OV3. Negligible change was noted in glioblastoma line U-87, breast cancer line MDA-MB-435, osteosarcoma line HOS, melanoma line SK-MEL5, colorectal cancer line COLO205, and prostate cancer line PC3. The cell lines least permissive to TPV replication were the glioblastoma (U-87) and melanoma (SK-MEL5) cell lines.

A New World Primate Deficient in Tetherin-Mediated Restriction of Human Immunodeficiency Virus Type 1

Human immunodeficiency virus type 1 (HIV-1) does not replicate in primary cells of New World primates. To better understand this restriction, we expressed owl monkey (Aotus nancymaae) CD4 and CXCR4 in the owl monkey kidney cell line, OMK. An HIV-1 variant modified to evade the owl monkey restriction factor TRIM-cyp replicated efficiently in these cells but could not replicate in primary A. nancymaae CD4-positive T cells. To understand this difference, we examined APOBEC3G and tetherin orthologs from OMK cells and primary A. nancymaae cells. We observed that OMK cells expressed substantially lower levels of APOBEC3G than did A. nancymaae cells. A. nancymaae, but not marmoset (Callithrix jacchus), APOBEC3G was partially downregulated by HIV-1 vif and reduced but did not abolish HIV-1 replication when stably expressed in OMK cells. The functional difference between A. nancymaae and marmoset APOBEC3Gs mapped to residue 128, previously shown to distinguish African green monkey from human APOBEC3G. We also characterized tetherin orthologs from OMK and A. nancymaae cells. The A. nancymaae tetherin ortholog, but not OMK tetherin, prevented HIV-1 release. Alteration of threonine 181 of OMK tetherin rescued its function and its efficient N glycosylation. All alleles of Aotus lemurinus griseimembra examined, but none of A. nancymaae or Aotus vociferans, encoded this nonfunctional tetherin ortholog. Our data indicate that HIV-1 replication in owl monkeys is not restricted at entry but can be limited by APOBEC3G and tetherin. Further, A. lemurinus griseimembra does not restrict HIV-1 replication via tetherin, a property likely useful for the study of tetherin-restricted viruses.

760. Overcoming Rhesus Macaque Endogenous Restriction Factors during HIV-1 Vector Transduction

Top of pageAbstract The ability to deliver genes into hematopoietic stem cells via HIV-1 derived vectors holds promise to investigate the pathobiology and therapeutic applications for infectious diseases. In vivo HIV-1 infection models are limited. The NOD/SCID-hu mouse has been an excellent model to study thymic function and HIV-1 thymic depletion. However, to better understand HIV-1 pathogenesis in humans, non-human primate models must be used. The rhesus macaque simian immunodeficiency virus (SIV) model recapitulates aspects of the pathology seen in human HIV-1 infection. Our laboratory has designed and previously described a CCR5 intrabody HIV-1 vector that efficiently prevents R5-tropic HIV-1 entry and provides transduced CD4+ T cells with a selective advantage during a dendritic cell-mediated infection. Unfortunately, transduction with a HIV-1 derived vector undergoes a post-entry restriction when introduced into rhesus macaque cells. The target for rhesus macaque endogenous restriction factors lies within the HIV-1 capsid protein, and may be due to rhesus macaque Cyclophilin A (CypA) incorporation and TRIM-5|[alpha]| capsid interactions. To increase transduction efficacy in both non-human primate and human hematopoietic cells we have assessed a naturally occurring HIV-1 capsid motif (V86P/H87Q/I91V/M96I), which evades both CypA- dependence in human cells and TRIM-5|[alpha]|-CypA restriction in owl monkey kidney cells during HIV-1 infection (Chatterji, et al. JBC, 2005). The V86P/H87Q/I91V/M96I capsid mutation within the CypA binding loop also rendered VSV-G pseudotyped HIV-1 vectors independent of CypA incorporation during human and non-human primate cell transduction. Interestingly, HIV-1 vectors with the V86P/H87Q/I91V/M96I capsid mutation were able to efficiently transduce non-human primate Vero and sMAGI cell lines and yielded a 1.4-fold increase in transduction efficiency in primary human CD4+ T cells. However, the V86P/H87Q/I91V/M96I HIV-1 vector did not efficiently transduce primary rhesus macaque CD4+ T cells. We are now in the process of evaluating novel HIV-1 capsid mutations that lie within and around the CypA binding loop in our HIV-1 vector system, such as H87Q and Q50Y/T54Q. Some of these capsid mutations in HIV-1 are required to efficiently infect primary rhesus macaque macrophages. We will present our findings on HIV-1 capsid mutations that allow HIV-1 vector entry into primary rhesus macaque CD4+ T cells.

368. Lentiviral Vector Strain of Origin and Target Cell Species-Specific Restriction Factors Influence Transduction Efficiency

The efficiency of transduction of both human and non-human primate cells by lentiviral vectors is influenced by interactions with members of the TRIM5|[alpha]| family of cytoplasmic proteins. This has been a particular problem when using HIV-1-derived vectors in rhesus macaque hematopoietic cells, where the vectors are strongly restricted by rhesus TRIM5|[alpha]|, although SIV-derived vectors are not. Current models to explain this variation in restriction postulate that the recruitment of cyclophilin A (CypA) by HIV-1 capsid protects the virus from TRIM5|[alpha]| restriction in human cells, but not in macaque cells. Conversely, although the SIVmac capsid does not bind CypA, it is not significantly restricted by human TRIM5|[alpha]|. We wanted to further investigate the role of the CypA-capsid interaction in the restriction of lentiviral vectors by transducing various cell types with VSV-G-pseudotyped lentiviral vectors expressing the enhanced green fluorescent protein (eGFP) gene and then analyzing gene expression by FACS. As expected, we found that treatment of human embryonic kidney cells (HEK293A) cells with cyclosporine A (CsA), which inhibits the CypA-capsid interaction, resulted in a |[sim]|2-fold drop in transduction with the HIV-1-based vector. Inclusion of either the HIV-2 or SIV nef proteins in the vector did not relieve its sensitivity to CsA. In contrast, transduction with HIV-1 vectors containing certain capsid substitutions within the CypA-binding site (Chatterji et al., JBC;280(48):40293|[ndash]|40300) was not negatively affected by CsA treatment, suggesting that the mutated vectors no longer require CypA for efficient transduction of human cells. These vectors also transduced owl monkey and rhesus macaque kidney cells more efficiently than the wild-type vector. Vectors derived from different SIVmac-strains were found to transduce rhesus macaque cell lines at equal efficiency, but transduction of primary rhesus CD34+ cells with an SIVmac1A11-derived vector (Hanawa et al., Blood;103:4062|[ndash]|4069) was significantly more efficient (30|[ndash]|60 %) than with an SIVmac251-derived vector (4|[ndash]|12%) (N|[egrave]|gre et al., Gene Therapy;7:1613|[ndash]|1623). Experiments in which the SIVmac transfer vectors were crosspackaged with capsids from the other SIVmac strains revealed that this property maps to the transfer vector and not to the packaging construct. CsA treatment increased transduction with SIVmac-derived vectors in both rhesus and owl monkey cell lines and in primary human CD34+ cells, suggesting that the SIVmac capsid is susceptible to restriction in these tissues. In contrast, CsA treatment had no effect on transduction with either HIV-1 or SIVmac-derived vectors in primary rhesus macaque CD34+ cells. Together, these results show differential restriction of lentiviral vectors that varies with the species and cell type transduced. These findings may be relevant for studies using lentiviral gene transfer into non-human primate models.

A Cyclophilin A-Independent Capsid Motif Renders HIV-1 Derived Vectors More Efficient at Transducing Human Cells.

We recently identified naturally occurring HIV-1 capsid (CA) motifs, which evade both cyclophilin A (CypA)-dependence in human cells and TRIM-5α-CypA (TRIM-Cyp) restriction in owl monkey kidney (OMK) cells (Chatterji, et al. JBC, In press). These HIV-1 capsid motifs contained either one (G89V) or four (V86P/H87Q/I91V/M96I) amino acid substitutions within the CypA binding site, but only the four amino acid mutant was able to bind CypA, yet does not require CypA incorporation to infect human cells. OMK cells are naturally resistant to HIV-1 infection due to TRIM-Cyp, a negative host factor, which efficiently targets incoming wild type HIV-1 capsid and thereby prevents the proper uncoating of the CA core. However, addition of cyclosporine A, which blocks the interaction of CypA and CA, or mutations within the CypA-CA binding site (G89V) yields OMK cells permissive to HIV-1. Interestingly, HIV-1 infection with the V86P/H87Q/I91V/M96I capsid mutant was able to achieve similar levels of infection in OMK cells as does wild type HIV-1 with the addition of cyclosporine A, emphasizing that this motif allows infection despite TRIM-Cyp-CA interactions. Alternatively, the interaction between HIV-1 CA and CypA, is considered as a positive host factor in human cells. It has been proposed that CypA either “protects” the virus from endogenous restriction factors or allows proper CA core uncoating. Human cells lacking CypA or treated with cyclosporine A are highly resistant to infection by wild type HIV-1. Virus is blocked during the early stage of infection, at the pre-reverse transcription step. An important goal of human gene therapy is to efficiently deliver genes to non-dividing or quiescent cells such as hematopoietic stem cells and T-cells, respectively. Improvements on vector design such as self-inactivating LTRs, VSV-G envelope pseudotyping, various viral capsids, and enhancer elements have improved both the safety and efficiency of gene delivery. To identify capsid motifs that enhance transduction, we assessed whether the G89V and V86P/H87Q/I91V/M96I capsid motifs increased transduction efficacy in primary human cells as compared to vectors packaged with wild type HIV-1 gag/pol. Preliminary studies, at low levels of transduction (2000 pg p24 input ~1 MOI) in 293T cells, showed a 3-fold increase in GFP expression using the V86P/H87Q/I91V/M96I-gag/pol packaged vectors as compared to wild type or G89V-gag/pol vectors. Studies with primary PBMC and CD34+ hematopoietic stem cells are underway and will address whether the V86P/H87Q/I91V/M96I capsid motif will allow for greater transduction efficiency for non-cycling, quiescent and/or partially activated cells. Providing a means to enhance uniform vector transduction efficacy should allow for lower levels of vector input during transduction, thereby reducing the probability for insertional mutagenesis.

A Trim5-cyclophilin A fusion protein found in owl monkey kidney cells can restrict HIV-1.

Lv1 restriction of HIV-1 in the cells of Old World monkeys is associated with the expression of the Trim5 gene. Uniquely, in owl monkey kidney cells, HIV-1 restriction is dependent on the ability of incoming viral capsid protein to bind cyclophilin A (CypA). Cloning of the owl monkey Trim5 gene now reveals the presence of an inserted CypA pseudogene within intron 7 of the Trim5 gene. This insertion results in the formation of a chimeric Trim5-CypA transcript. Transfer of a cDNA corresponding to this transcript into human cells confers cyclosporin A-sensitive resistance to HIV-1 infection. The restriction factor appears to be a chimeric protein created by retrotransposon-mediated exon shuffling.

The replication cycle of tanapox virus in owl monkey kidney cells

The growth kinetics of tanapox virus in owl monkey kidney cells was elucidated by single-step growth curves at multiplicities of 10, 1.0, and 0.1 plaque forming units (pfu) per cell at 37 and 33°C. Virus replicated equally well at both temperatures and produced a cytopathic effect that was characterized by densely packed rounded cells with retrogressed monolayer and granular vacuolated cytoplasm. Single-step growth curves revealed that the eclipse period varied from 24 h postinfection (hpi) at a multiplicity of infection of 10 pfu/cell to 48 hpi at 0.1 pfu/cell. The length of the latent period also varied from 36 hpi at 10 pfu/cell to 48 hpi at 0.1 pfu/cell. The intracellular virus, extracellular virus, and total virus titers reached their maximums relatively early at 10 pfu/cell as compared with 0.1 pfu/cell. About 78% of the mature progeny virion is retained intracellularly at 10 pfu/cell at 96 hpi. We conclude that tanapox virus replication is similar to other poxviruses, but the replication cycle is longer when compared with vaccinia virus.Key words: tanapox virus, single-step growth curve, eclipse period, latent period.

The replication cycle of tanapox virus in owl monkey kidney cells.

The growth kinetics of tanapox virus in owl monkey kidney cells was elucidated by single-step growth curves at multiplicities of 10, 1.0, and 0.1 plaque forming units (pfu) per cell at 37 and 33 degrees C. Virus replicated equally well at both temperatures and produced a cytopathic effect that was characterized by densely packed rounded cells with retrogressed monolayer and granular vacuolated cytoplasm. Single-step growth curves revealed that the eclipse period varied from 24 h postinfection (hpi) at a multiplicity of infection of 10 pfu/cell to 48 hpi at 0.1 pfu/cell. The length of the latent period also varied from 36 hpi at 10 pfu/cell to 48 hpi at 0.1 pfu/cell. The intracellular virus, extracellular virus, and total virus titers reached their maximums relatively early at 10 pfu/cell as compared with 0.1 pfu/cell. About 78% of the mature progeny virion is retained intracellularly at 10 pfu/cell at 96 hpi. We conclude that tanapox virus replication is similar to other poxviruses, but the replication cycle is longer when compared with vaccinia virus.

A single amino acid change in the hemagglutinin protein of measles virus determines its ability to bind CD46 and reveals another receptor on marmoset B cells.

This paper provides evidence for a measles virus receptor other than CD46 on transformed marmoset and human B cells. We first showed that most tissues of marmosets are missing the SCR1 domain of CD46, which is essential for the binding of Edmonston measles virus, a laboratory strain that has been propagated in Vero monkey kidney cells. In spite of this deletion, the common marmoset was shown to be susceptible to infections by wild-type isolates of measles virus, although they did not support Edmonston measles virus production. As one would expect from these results, measles virus could not be propagated in owl monkey or marmoset kidney cell lines, but surprisingly, both a wild-type isolate (Montefiore 89) and the Edmonston laboratory strain of measles virus grew efficiently in B95-8 marmoset B cells. In addition, antibodies directed against CD46 had no effect on wild-type infections of marmoset B cells and only partially inhibited the replication of the Edmonston laboratory strain in the same cells. A direct binding assay with insect cells expressing the hemagglutinin (H) proteins of either the Edmonston or Montefiore 89 measles virus strains was used to probe the receptors on these B cells. Insect cells expressing Edmonston H but not the wild-type H bound to rodent cells with CD46 on their surface. On the other hand, both the Montefiore 89 H and Edmonston H proteins adhered to marmoset and human B cells. Most wild-type H proteins have asparagine residues at position 481 and can be converted to a CD46-binding phenotype by replacement of the residue with tyrosine. Similarly, the Edmonston H protein did not bind CD46 when its Tyr481 was converted to asparagine. However, this mutation did not affect the ability of Edmonston H to bind marmoset and human B cells. The preceding results provide evidence, through the use of a direct binding assay, that a second receptor for measles virus is present on primate B cells.

Anti-apoptotic bcl-2 homologs encoded by Kaposi's sarcoma-associated virus and herpesvirus saimiri; a role during virus infections

Background: Cellular bcl-2 was identified at t(14;18) translocation breakpoints in follicular lymphomas and is a potent inhibitor of programmed cell death induced by a variety of death stimuli. Bcl-2 is a 26kD mitochondrial membrane protein that contributes to tumorigenesis by preventing cell death rather than inducing cell proliferation and can complement c-myc in cell transformation. We have identified homologs of the cellular bcl-2 oncogene encoded by the gamma herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and herpesvirus saimiri(HVS). HVS is a T-cell tropic virus that causes rapid fatal lymphomas in monkeys. However, the role of HVS and KSHV viral bcl-2 homologs during infection or tumorigenesis is not known. Methods: The function of viral bcl-2 homologs was assessed by using a Sindbis virus vector system to express viral homologs of bcl-2. Recombinant HVS deleted forbcl-2 was constructed by homologous recombination. Results: Overexpression of KSbcl-2 of KSHV and ORF 16 (bcl-2 homolog) of HVS blocked apoptosis as efficiently as Bcl-2, Bcl-xL or another viral Bcl-2 homolog encoded by Epstein-Barr virus (BHRF1). Interestingly, KSbcl-2 neither homodimerizes nor heterodimerizes with other Bcl-2 family members, suggesting that KSbcl-2 may have evolved to escape the negative regulatory effects of the proapoptotic cellular homologs Bax and Bak. Furthermore, the herpesvirus Bcl-2 homologs including KSbcl-2, BHRF1, and ORF16 contain poorly conserved Bcl-2 homology 3 (BH3) domains compared to mammalian Bcl-2 homologs, implying that BH3 may not be essential for anti-apoptotic function. Consistent with the biology of Epstein-Barr virus infections, KSbcl-2 appears to be expressed during the lytic phase of the viral replication cycle. To begin to assess the role of herpesvirus bcl-2 homologs in viral replication and tumorigenesis, the bcl-w homolog of HVS was replaced with ßGAL. The resulting recombinant viruses exhibited a null plaque phenotype. Conclusions: Although bcl-2 homolog-deficient Epstein-Barr virus lacks an observable phenotype, bcl-2 homolog-deficient HVS was unable to produce plaques on Owl Monkey Kidney cells, suggesting that the anti-apoptotic function was required for efficient virus replication. KSbcl-2 of KSHV may have a similar role.

Antigenic and genomic identity between simian herpesvirus aotus type 2 and bovine herpesvirus type 4

Herpesvirus aotus type 2 (HVA-2) was isolated from a culture of kidney cells from a healthy owl monkey (Aotus trivirgatus). Bovine herpesvirus type 4 (BHV-4) is frequently isolated from diseased and even healthy cattle and occasionally from sheep, wild ruminants and cats. The two viruses are related antigenically, as was revealed by an indirect fluorescent antibody test using polyclonal antisera from experimentally infected rabbits or monoclonal antibodies raised against six BHV-4 proteins, three of which were glycosylated. The genome structures of the two viruses consist of a unique central sequence flanked at both ends by G + C-rich tandem repeats. Restriction maps (produced using EcoRI, BamHI and HindIII) of these two viruses were nearly identical but the unique sequence of the HVA-2 genome possessed two additional BamHI sites. Four genomic regions of variable size were detected, two located in the unique part, one in the repetitive part and one in the left junction between the unique and the repeated part of the genome; these slight variations were similar to those observed between various BHV-4 isolates. These results suggest that HVA-2 and BHV-4 belong to the same virus species; HVA-2 could be either a BHV-4 contaminant of owl monkey kidney cell cultures or an isolate from an owl monkey accidentally infected with BHV-4.

Trans activation of the thymidylate synthase promoter of herpesvirus saimiri

Herpesvirus saimiri has been shown to possess a thymidylate synthase (TS) gene that is unusual in its transcriptional regulation. Although TS is believed to be required for viral DNA synthesis, the TS-specific 2.5-kb mRNA was found most abundantly during the late phases of asynchronous virus replication in permissive cultures. To study the kinetics of gene activation, the TS promoter and regulatory sequences were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene. No CAT expression or transcripts were found after transfection of fusion genes into permissive owl monkey kidney (OMK) cells. However, the promoter was strongly activated when CAT plasmids were cotransfected with intact herpesvirus saimiri virion DNA or were transferred to OMK cells that were lytically infected with herpesvirus saimiri or a related herpesvirus, herpesvirus ateles. CAT was expressed at reduced levels in cultures when viral DNA replication was inhibited by phosphonoacetic acid; this indicates that the gene is activated during the delayed-early phase. However, the highest amounts of mRNA were present in the late period of replication. Deletion analyses localized essential response elements for trans activation in the promoter upstream region between nucleotides -311 and -56; they consisted of related tandem repeats and perfect palindromes. A sequence with two overlapping palindromes of 16 and 18 bp was found to be a major target for activation of the herpesvirus saimiri TS promoter. These palindromes did not have any significant homologies with known sequences of herpesviruses or cellular DNA; the 18-bp palindrome had, however, a certain structural similarity with a conserved sequence of the E2-responsive cis sequence that is required for transcription regulation of early papillomavirus genes.

A comparison of BKV, JCV, and SV 40 transcriptional enhancers in primate cells

The activities of the transcriptional enhancers of the prototype human polyomaviruses, BKV (Dun) and JCV (MAD-1), were compared with that of the rhesus virus SV 40 in Old World African green monkey kidney cells (CV-1) and in New World owl monkey kidney cells (OMK). Enhancer activities were tested in CAT-expression plasmids. CAT activity in transfected cell extracts were measured by the novel two-phase partition assay. The activities of the BKV and JCV enhancers were 45% and 20% that of SV 40, respectively, in CV-1 cells. The effectiveness of each viral enhancer was less in OMK cells than in CV-1 cells, as would be expected if the viruses co-evolved with their natural hosts. In CV-1 cells, the BKV enhancer was more active than that of JCV, but in OMK cells the reverse was true. This result is significant in view of the fact that JCV, but not BKV, is oncogenic in owl monkeys.

Synthesis of bovine growth hormone in primates by using a herpesvirus vector.

A strain of herpesvirus saimiri containing a bovine growth hormone (bGH) gene under the control of the simian virus 40 (SV40) late-region promoter was constructed. This strain, bGH-Z20, was replication competent and stably harbored the bGH gene upon serial passage. Nonpermissive marmoset T cells persistently infected with bGH-Z20 produced a 0.9-kilobase RNA which contained all of the bGH exon sequences and appeared to initiate within the SV40 promoter region. However, in permissively infected owl monkey kidney cells, RNAs containing growth hormone sequences appeared to initiate from herpesvirus saimiri promoters positioned upstream from the SV40-growth hormone gene. Persistently infected T cells in culture secreted 500 ng of bGH protein per 10(6) cells per 24 h during the several months of testing. The secreted protein was 21 kilodaltons, the size of authentic bGH. New World primates experimentally infected with bGH-Z20 produced circulating bGH and developed immunoglobulin G antibodies directed against bGH. Because herpesviruses characteristically remain latent in the infected host, these observations suggest a means for replacing gene products missing or defective in hereditary genetic disorders.

Synthesis of bovine growth hormone in primates by using a herpesvirus vector.

A strain of herpesvirus saimiri containing a bovine growth hormone (bGH) gene under the control of the simian virus 40 (SV40) late-region promoter was constructed. This strain, bGH-Z20, was replication competent and stably harbored the bGH gene upon serial passage. Nonpermissive marmoset T cells persistently infected with bGH-Z20 produced a 0.9-kilobase RNA which contained all of the bGH exon sequences and appeared to initiate within the SV40 promoter region. However, in permissively infected owl monkey kidney cells, RNAs containing growth hormone sequences appeared to initiate from herpesvirus saimiri promoters positioned upstream from the SV40-growth hormone gene. Persistently infected T cells in culture secreted 500 ng of bGH protein per 10(6) cells per 24 h during the several months of testing. The secreted protein was 21 kilodaltons, the size of authentic bGH. New World primates experimentally infected with bGH-Z20 produced circulating bGH and developed immunoglobulin G antibodies directed against bGH. Because herpesviruses characteristically remain latent in the infected host, these observations suggest a means for replacing gene products missing or defective in hereditary genetic disorders.

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.

A single major immediate-early virus gene product is synthesized in cells productively infected with herpesvirus saimiri.

A major virus-specific immediate-early (IE) polypeptide with an apparent mol. wt. of 52 000 (52K) was synthesized immediately after the removal of a cycloheximide block applied to owl monkey kidney cell cultures at the time of infection with high multiplicities of herpesvirus saimiri (HVS). The IE 52K polypeptide was phosphorylated and accumulated rapidly and efficiently in the nucleus of infected cells. Monoclonal antibodies to IE 52K and to a major non-structural delayed-early (DE) DNA-binding protein with similar electrophoretic mobility (DE 51K) were used to show that the IE 52K protein and DE 51K proteins are antigenically distinct. The monoclonal antibody to the IE 52K protein of HVS strain 11 reacted in immune precipitation and immunofluorescence tests with polypeptides of similar mol. wt. present as nuclear antigens in cells infected with heterologous strains of HVS and herpesvirus ateles.