Analysis of Simian Endogenous Retrovirus (SERV) Full-Length Proviruses in Old World Monkey Genomes.

Simian endogenous retrovirus, SERV, is a successful germ line invader restricted to Old World monkey (OWM) species. (1) Background: The availability of high-quality primate genomes warrants a study of the characteristics, evolution, and distribution of SERV proviruses. (2) Methods: Cercopithecinae OWM genomes from public databases were queried for the presence of full-length SERV proviruses. A dataset of 81 Cer-SERV genomes was generated and analyzed. (3) Results: Full-length Cer-SERV proviruses were mainly found in terrestrial OWM, and less so in arboreal, forest- dwelling monkeys. Phylogenetic analysis confirmed the existence of two genotypes, Cer-SERV-1 and Cer-SERV-2, with Cer-SERV-1 showing evidence of recent germ-line expansions. Long Terminal Repeat (LTR) variation indicated that most proviruses were of a similar age and were estimated to be between <0.3 and 10 million years old. Integrations shared between species were relatively rare. Sequence analysis further showed extensive CpG methylation-associated mutations, variable Primer Binding Site (PBS) use with Cer-SERV-1 using PBSlys3 and Cer-SERV-2 using PBSlys1,2, and the recent gain of LTR motifs for transcription factors active during embryogenesis in Cer-SERV-1. (4) Conclusions: sequence analysis of 81 SERV proviruses from Cercopithecinae OWM genomes provides evidence for the adaptation of this retrovirus to germ line reproduction.

Novel endogenous simian retroviral integrations in Vero cells: implications for quality control of a human vaccine cell substrate.

African green monkey (AGM)-derived Vero cells have been utilized to produce various human vaccines. The Vero cell genome harbors a variety of simian endogenous type D retrovirus (SERV) sequences. In this study, a transcriptome analysis showed that DNA hypomethylation released the epigenetic repression of SERVs in Vero cells. Moreover, comparative genomic analysis of three Vero cell sublines and an AGM reference revealed that the genomes of the sublines have ~80 SERV integrations. Among them, ~60 integrations are present within all three cell sublines and absent from the reference sequence. At least several of these integrations consist of complete SERV proviruses. These results strongly suggest that SERVs integrated in the genome of Vero cells did not retrotranspose after the establishment of the cell lineage as far as cells were maintained under standard culture and passage conditions, providing a scientific basis for controlling the quality of pharmaceutical cell substrates and their derived biologics.

Mucosal Vaccination with Heterologous Viral Vectored Vaccine Targeting Subdominant SIV Accessory Antigens Strongly Inhibits Early Viral Replication.

Conventional HIV T cell vaccine strategies have not been successful in containing acute peak viremia, nor in providing long-term control. We immunized rhesus macaques intramuscularly and rectally using a heterologous adenovirus vectored SIV vaccine regimen encoding normally weakly immunogenic tat, vif, rev and vpr antigens fused to the MHC class II associated invariant chain. Immunizations induced broad T cell responses in all vaccinees. Following up to 10 repeated low-dose intrarectal challenges, vaccinees suppressed early viral replication (P=0.01) and prevented the peak viremia in 5/6 animals. Despite consistently undetectable viremia in 2 out of 6 vaccinees, all animals showed evidence of infection induced immune responses indicating that infection had taken place. Vaccinees, with and without detectable viremia better preserved their rectal CD4+ T cell population and had reduced immune hyperactivation as measured by naïve T cell depletion, Ki-67 and PD-1 expression on T cells. These results indicate that vaccination towards SIV accessory antigens vaccine can provide a level of acute control of SIV replication with a suggestion of beneficial immunological consequences in infected animals of unknown long-term significance. In conclusion, our studies demonstrate that a vaccine encoding subdominant antigens not normally associated with virus control can exert a significant impact on acute peak viremia.

Roles of the three L-domains in ß-retrovirus budding.

Retroviral Gag protein plays a critical role during the late stage of virus budding and possesses a so-called L-domain containing PT/SAP, PPxY, YxxL or FPIV motifs that are critical for efficient budding. Mason-Pfizer monkey virus (M-PMV) contains PSAP, PPPY, and YADL sequences in Gag. This study was performed to investigate the roles of these three L-domain-like sequences in virus replication in three different cell lines, 293T, COS-7 and HeLa cells. It was found that the PPxY motif plays an essential role in progeny virus production as a major L-domain in all three cell lines. The PSAP sequence was shown to function as an additional L-domain in HeLa cells and to promote efficient release of M-PMV; however, this sequence was dispensable for M-PMV production in 293T and COS-7 cells, suggesting that the role of the PSAP motif as an L-domain in M-PMV budding is cell type-dependent. Viruses possessing multiple L-domains appear to change the L-domain usage to replicate in various cells. On the other hand, the YADL motif was required for M-PMV production as a transport signal of Gag to the plasma membrane, but not as an L-domain.

Virological and serological characterization of SRV-4 infection in cynomolgus macaques.

The nature of SRV-4 infection in cynomolgus macaques remains unclear to date. Here, we report the monitoring of 24 cynomolgus monkeys that were naturally infected with SRV-4 for virus isolation, proviral load and antibody. The results indicated that the SRV-4 antibody status was statistically correlated to environmental temperature.

Simian retroviruses: infection and disease--implications for immunotoxicology research in primates.

Non-human primates have assumed an important role in preclinical safety assessment studies, particularly in the evaluation of biopharmaceutical and immunomodulatory therapies. Naturally occurring simian retrovirus infections may adversely affect the suitability of primates for use in such studies. Various species of non-human primates are the natural hosts for six exogenous retroviruses, representing five genera within the family Retroviridae. Retroviruses establish persistent infections with a broad spectrum of pathogenic potential, ranging from nonpathogenic to highly pathogenic, depending on the variety of the host, virus, and environmental factors. In the context of immunotoxicology, in which the research objective is to specifically evaluate the effect of drugs or biologics on the immune system, the immune modulatory effects of simian retroviruses, which may be subtle or profound, may introduce significant confounding into the studies of immunotoxic effects utilizing non-human primates. Latent or subclinical retrovirus infections are common and research-related procedures may lead to virus reactivation or overt disease. Adverse effects of undetected retrovirus infections on preclinical research include the loss of experimental subjects (and potentially of statistical power) due to increased morbidity and mortality, virus-induced clinical abnormalities, histologic lesions, alteration of physiologic parameters and biologic responses, and interference with in vitro assays and/or cytolytic destruction of primary cell cultures. The aim of this review is to provide an overview of the key biological, clinical, and pathological features of several important simian retroviruses, with emphasis on viruses infecting macaques and other primate species commonly used in preclinical research, and a discussion of the implications of these infections for immunotoxicology and other preclinical research in primates. Adequate pre-study retrovirus screening is essential to exclude retrovirus-infected primates from research protocols.

An updated review of simian betaretrovirus (SRV) in macaque hosts.

This review is an updated summary of nearly 30 years of SRV history and provides new and critical findings of original research accomplished in the last 5 years including, but not limited to, the pathogenetic mechanisms underlying the origin of hematopoietic abnormalities observed in infected hosts and proposed new SRV serotypes. Despite major advances in the understanding and control of SRV disease, much more remains to be learned and SRV continues to be an exciting and attractive primate model for comparative studies of the mechanisms of retroviral immunosuppression.

Detection of SRV/D shedding in body fluids of cynomolgus macaques and comparison of partial gp70 sequences in SRV/D-T isolates.

We previously reported the isolation of a novel subtype of SRV/D-Tsukuba (SRV/D-T) from two cynomolgus monkeys (Macaca facicularis) in the breeding colony of Tsukuba Primate Research Center (TPRC). We surveyed for SRV/D infection in the TPRC cynomolgus colony using SRV/D-specific PCR primer sets designed based on the entire gag region sequence. The only SRV/D subtype detected in the colony was SRV/D-T with a positive infection rate of 22.4% (n = 49). It has been reported that the mode of transmission of SRV/D is via contact with virus shed in the body fluids. In this report, to investigate the infection route of SRV/D-T in monkeys at TPRC, we performed virus isolation and PCR for detection of the SRV/D genome from peripheral blood mononuclear cells (PBMCs), plasma, saliva, urine, and feces. Virus isolation and PCR detection were positive in plasma, saliva, urine, and fecal samples from all monkeys on which virus was isolated from PBMCs. This suggests that the spread of SRV/D-T infection in TPRC is via contact with virus shed in saliva, urine, and/or feces. Also, comparison of sequences of gp70 on multiple SRV/D-T isolates revealed that there was little intra- and inter-monkey variation, suggesting that SRV/D-T is fairly stable.

Increased viral replication in simian immunodeficiency virus/simian-HIV-infected macaques with self-administering model of chronic alcohol consumption.

Alcohol abuse constitutes a major cohort among HIV-infected individuals. The precise effect of alcohol addiction on HIV pathogenesis remains inconclusive, however. This study was designed to determine the effect of alcohol dependence on virus replication and CD4 profiles in simian immunodeficiency virus/simian-HIV-infected rhesus macaques. A group of 3 male Indian rhesus macaques was adapted to a self-drinking model of alcohol consumption, whereas another group of 3 macaques was provided a Nutrasweet solution. After 7 weeks of alcohol consumption, the alcohol-dependent animals along with controls were intravenously inoculated with a mixture of SHIV(KU), SHIV(89.6)P, and SIV/17E-Fr. These animals were followed for a period of 24 weeks for complete blood cell counts, CD4 cell profiles, and viral loads in the blood and cerebral compartments. The alcohol and control groups showed comparable peak viral loads in the blood. The plasma viral load in the alcohol group was 31- to 85-fold higher than that in the control group at weeks 18 through 24 after infection, however. The pattern of cerebrospinal fluid viral replication was also comparable during the acute phase; however, the virus continued to replicate in the brain of alcohol-dependent animals, whereas it became undetectable in the controls. The extent of CD4 cell loss in the alcohol group was significantly higher than that in the control animals at week 1 after infection.

Virus load and sequence variation in simian retrovirus type 2 infection.

The natural history of type D simian retrovirus (SRV) infection is poorly characterized in terms of viral load, antibody status, and sequence variation. To investigate this, blood samples were taken from a small cohort of mostly asymptomatic cynomolgus macaques (Macaca fascicularis), naturally infected with SRV type 2 (SRV-2), some of which were followed over an 8-month period with blood taken every 2 months. Provirus and RNA virus loads were obtained, the samples were screened for presence of antibodies to SRV-2 and neutralizing antibody titers to SRV-2 were assayed. env sequences were aligned to determine intra- and intermonkey variation over time. Virus loads varied greatly among cohort individuals but, conversely, remained steady for each macaque over the 8-month period, regardless of their initial levels. No significant sequence variation was found within an individual over time. No clear picture emerged from these results, which indicate that the variables of SRV-2 infection are complex, differ from those for lentivirus infection, and are not distinctly related to disease outcome.

Microglial activation and neurological symptoms in the SIV model of NeuroAIDS: association of MHC-II and MMP-9 expression with behavioral deficits and evoked potential changes.

HIV-1 causes cognitive and motor deficits and HIV encephalitis (HIVE) in a significant proportion of AIDS patients. Neurological impairment and HIVE are thought to result from release of cytokines and other harmful substances from infected, activated microglia. In this study, the quantitative relationship between microglial activation and neurological impairment was examined in the simian immunodeficiency model of HIVE. Macaque monkeys were infected with a passaged, neurovirulent strain of simian immunodeficiency virus, SIV(mac)239(R71/17E). In concurrent studies, functional impairment was assessed by motor and auditory brainstem evoked potentials and by measurements of cognitive and motor behavioral deficits. Brain tissue was examined by immunohistochemistry using two markers of microglia activation, MHC-II and matrix metalloproteinase-9 (MMP-9). The inoculated animals formed two groups: rapid progressors, which survived 6-14 weeks postinoculation, and slow progressors, which survived 87-109 weeks. In the rapid progressors, two patterns of MHC-II expression were present: (1) a widely disseminated pattern of MHC-II expressing microglia and microglial nodules in cortical gray matter and subcortical white matter, and (2) a more focal pattern in which MHC-II expressing microglia were concentrated into white matter. Animals exhibiting both patterns of microglial activation showed mild to severe changes in cognitive and motor behavior and evoked potentials. All rapid progressors showed expression of MMP-9 in microglia located in subcortical white matter. In the slow progressors MHC-II and MMP-9 staining was similar to uninoculated control macaques, and there was little or no evidence of HIVE. These animals showed behavioral deficits at the end of the disease course, but little changes in evoked potentials. Thus, increases in MHC-II and MMP-9 expression are associated with development of cognitive and motor deficits, alterations in evoked potentials, and rapid disease progression.

A sodium-dependent neutral-amino-acid transporter mediates infections of feline and baboon endogenous retroviruses and simian type D retroviruses.

The type D simian retroviruses cause immunosuppression in macaques and have been reported as a presumptive opportunistic infection in a patient with AIDS. Previous evidence based on viral interference has strongly suggested that the type D simian viruses share a common but unknown cell surface receptor with three type C viruses: feline endogenous virus (RD114), baboon endogenous virus, and avian reticuloendotheliosis virus. Furthermore, the receptor gene for these viruses has been mapped to human chromosome 19q13.1-13.2. We now report the isolation and characterization of a cell surface receptor for this group of retroviruses by using a human T-lymphocyte cDNA library in a retroviral vector. Swiss mouse fibroblasts (NIH 3T3), which are naturally resistant to RD114, were transduced with the retroviral library and then challenged with an RD114-pseudotyped virus containing a dominant selectable gene for puromycin resistance. Puromycin selection yielded 12 cellular clones that were highly susceptible to a beta-galactosidase-encoding lacZ(RD114) pseudotype virus. Using PCR primers specific for vector sequences, we amplified a common 2.9-kb product from 10 positive clones. Expression of the 2.9-kb cDNA in Chinese hamster ovary cells conferred susceptibility to RD114, baboon endogenous virus, and the type D simian retroviruses. The 2.9-kb cDNA predicted a protein of 541 amino acids that had 98% identity with the previously cloned human Na+-dependent neutral-amino-acid transporter Bo. Accordingly, expression of the RD114 receptor in NIH 3T3 cells resulted in enhanced cellular uptake of L-[3H]alanine and L-[3H]glutamine. RNA blot (Northern) analysis suggested that the RD114 receptor is widely expressed in human tissues and cell lines, including hematopoietic cells. The human Bo transporter gene has been previously mapped to 19q13.3, which is closely linked to the gene locus of the RD114 receptor.

The RD114/simian type D retrovirus receptor is a neutral amino acid transporter.

The RD114/simian type D retroviruses, which include the feline endogenous retrovirus RD114, all strains of simian immunosuppressive type D retroviruses, the avian reticuloendotheliosis group including spleen necrosis virus, and baboon endogenous virus, use a common cell-surface receptor for cell entry. We have used a retroviral cDNA library approach, involving transfer and expression of cDNAs from highly infectable HeLa cells to nonpermissive NIH 3T3 mouse cells, to clone and identify this receptor. The cloned cDNA, denoted RDR, is an allele of the previously cloned neutral amino acid transporter ATB0 (SLC1A5). Both RDR and ATB0 serve as retrovirus receptors and both show specific transport of neutral amino acids. We have localized the receptor by radiation hybrid mapping to a region of about 500-kb pairs on the long arm of human chromosome 19 at q13.3. Infection of cells with RD114/type D retroviruses results in impaired amino acid transport, suggesting a mechanism for virus toxicity and immunosuppression. The identification and functional characterization of this retrovirus receptor provide insight into the retrovirus life cycle and pathogenesis and will be an important tool for optimization of gene therapy using vectors derived from RD114/type D retroviruses.

Activation in vivo of retroperitoneal fibromatosis-associated herpesvirus, a simian homologue of human herpesvirus-8.

Retroperitoneal fibromatosis-associated herpesvirus of rhesus macaques (RFHVMm) is a gammaherpesvirus closely related to human herpesvirus-8 (HHV-8), which is thought to be a necessary cofactor for the development of Kaposi's sarcoma (KS) in humans. Here, RFHVMm infection of rhesus macaques exposed to the D-type retrovirus simian retrovirus-2 (SRV-2) is described. Development of SRV-2 viraemia, infection with simian immunodeficiency virus or administration of cyclosporin A could result in persistent RFHVMm viraemia. From this, it is concluded that productive retrovirus infection or otherwise-induced immune suppression has the ability to activate this herpesvirus in vivo. Elevated levels of circulating interleukin-6, a cytokine that plays a central role in KS, were found in RFHVMm-viraemic animals. In viraemic animals, RFHVMm was found in tissues that are common sites for the development of AIDS-associated KS, especially the oral cavity. Together, these data suggest a common biology between RFHVMm infection of macaques and HHV-8 infection and pathogenesis in humans.

The posttranscriptional control element of the simian retrovirus type 1 forms an extensive RNA secondary structure necessary for its function.

It was previously shown that a 240-nucleotide (nt) RNA element (cis-acting transactivation element [CTE]) located between the env gene and the 3' long terminal repeat of simian retrovirus type 1 (SRV-1) can functionally replace posttranscriptional activation directed by Rev and the Rev-responsive element (RRE) when inserted into a Rev- and RRE-deficient molecular clone of human immunodeficiency virus type 1, resulting in efficient virus replication. Here, we analyze the molecular and structural requirements for function of this RNA element. Deletion mutagenesis demonstrated that the core element spans 173 nt. SRV-2 and Mason-Pfizer monkey virus have highly homologous elements, which function similarly when inserted into the Rev/RRE-deficient human immunodeficiency virus type 1. Computer prediction indicated that the core CTEs of all three viruses have similar extensive secondary structures. Mutagenesis of the SRV-1 CTE revealed that both sequence and secondary structure are essential for function. Nuclease probing of the SRV-1 CTE further supported the genetic analysis and confirmed the predicted structural features of the RNA element. Sequence analysis of the 240-nt SRV-1 CTE, after continuous long-term propagation of the Rev-independent viruses, revealed that the genetically defined core element remained unchanged, while regions outside the core CTE underwent deletions or duplications. These data further support our in vitro mutagenesis data and demonstrate the importance of the sequence and structure of the SRV-1 CTE for appropriate function.

Simian AIDS type D serogroup 2 retrovirus: isolation of an infectious molecular clone and sequence analyses of its envelope glycoprotein gene and 3' long terminal repeat.

We describe the molecular cloning of a serogroup 2 simian retrovirus (SRV; D2/RHE/OR) and present the sequence of its envelope (env) glycoprotein gene and 3' long terminal repeat region. This report documents the first infectious molecular clone of a serogroup 2 SRV and provides env sequence verification of genetic diversity among serogroup 2 SRV isolates.

Pseudotyped REV/SRV retroviruses reveal restrictions to infection and host range within members of the same receptor interference group.

The reticuloendotheliosis virus (REV) group of retroviruses and type D simian retroviruses (SRV) belong to the same receptor interference group. The cellular receptor for these viruses has not yet been identified. In order to study the distribution of the receptor and to identify a receptor negative cell line, vector viral pseudotypes between REV and SRV were made. Using these viral pseudotypes, susceptibility to infection was examined in some rodent and marsupial cell lines. Infectivity assays with these envelope pseudotypes demonstrated that all cell types tested were resistant to infection. However, treatment of the rodent cells with the N-linked glycosylation inhibitor tunicamycin rendered most of the cells susceptible to infection. These results indicate that all the rodent cells tested express a nonfunctional receptor for viruses of the REV and SRV groups, which can be made functional by tunicamycin treatment. A difference in receptor host range among members of the same receptor interference group was observed which suggests that the REV/SRV receptor for this interference group might be differentially modified in different cell types. The studies also identified at least one cell line not expressing the REV/SRV receptor which should be useful for isolating the gene encoding the receptor.

Inhibitors of retrovirus infection are secreted by several hamster cell lines and are also present in hamster sera.

We have previously shown that Chinese hamster ovary (CHO) cells are resistant to infection by gibbon ape leukemia virus and amphotropic pseudotype retroviral vectors because of the secretion of factors that inhibit retrovirus infection. Such factors were not secreted by any mouse or human cell lines tested. Secretion of the inhibitors and resistance to infection are abrogated by treatment of CHO cells with the glycosylation inhibitor tunicamycin. Here we show that the inhibitory activities against gibbon ape leukemia virus and amphotropic viruses are partially separable and that glycosylation mutations in CHO cells mimic the effects of tunicamycin treatment. We find that several hamster cell lines derived from both Chinese and Syrian hamsters secrete inhibitors of retrovirus infection, showing that these inhibitors are not unique to the CHO cell line. Inhibitory factors are also present in the sera of Chinese and Syrian hamsters but were not detected in bovine serum. These results suggest the presence of specific factors that function to inhibit retrovirus infection in hamsters.