Experimental transmission of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) to SIV-positive and SIV-negative rhesus macaques.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gamma-herpesvirus associated with Kaposi's sarcoma (KS) and two lymphoproliferative diseases, primary effusion lymphoma (PEL) and multicentric Castleman's disease. Studies on the biology and pathogenesis of KSHV have been limited by lack of efficient cell culture systems and lack of a suitable animal model for KS. Here we report on the experimental inoculation of SIV-positive and SIV-negative rhesus macaques with KSHV-infected PEL cells or KSHV preparations derived from PEL cells. Low levels of viral DNA could be detected in cultivated peripheral blood mononuclear cell of all animals, as well as in the bone marrow of one monkey that died from SAIDS. However, we were not able to detect KSHV-specific antibodies or transcripts, nor did we observe any symptoms clearly related to KSHV infection (e.g. KS or lympho-proliferative disease). Hence, although KSHV replicates in rhesus macaques at very low levels, this non-human primate host is unlikely to provide a useful animal model for disease.

Real-time TaqMan PCR as a specific and more sensitive alternative to the branched-chain DNA assay for quantitation of simian immunodeficiency virus RNA.

We developed a rapid and highly reproducible assay based on real-time PCR (TaqMan, Applied Biosystems, Foster City, CA) to quantitate simian immunodeficiency virus (SIV) RNA in plasma samples. This assay was compared with the current branched-chain DNA assay (Bayer, Emeryville, CA). Results obtained with the real-time TaqMan PCR assay were comparable to those obtained with the branched-chain DNA assay in overlapping ranges of sensitivities (r = 0.9429, p < 0.05). However, the real-time TaqMan PCR assay was capable of detecting as few as 50 copies of RNA/ml, whereas branched-chain DNA was only sensitive to 1,500 copies of RNA/ml. Therefore, several animals that tested negative by branched-chain DNA were positive by realtime TaqMan PCR. Two false positive tests were also recorded for the branched-chain DNA test. False negative and positive tests were confirmed by cell culture isolation and conventional nested RT-PCR. The SIV TaqMan assay detected a wide range of wild-type, cloned, and recombinant SIV strains with similar amplification efficiency, including SIVmac251, SIVmac239, SIVmac239 containing the 184V mutation in RT, SIV1A11, SIVmac239 delta3, SIVmac-M4, and chimeras (SHIVs) containing specific HIV-1 genes, such as reverse transcriptase (RT-SHIV) or Env (SHIV-E). In conclusion, the high sensitivity, increased specificity, wide dynamic range, simplicity, and reproducibility of the real-time SIV RNA quantitation allow the screening of large numbers of samples and make this method especially suitable for measuring both viral DNA and RNA levels during vaccine and therapy studies.

Simian-human immunodeficiency virus containing a human immunodeficiency virus type 1 subtype-E envelope gene: persistent infection, CD4(+) T-cell depletion, and mucosal membrane transmission in macaques.

The envelope (env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) determines several viral properties (e.g., coreceptor usage, cell tropism, and cytopathicity) and is a major target of antiviral immune responses. Most investigations on env have been conducted on subtype-B viral strains, prevalent in North America and Europe. Our study aimed to analyze env genes of subtype-E viral strains, prevalent in Asia and Africa, with a nonhuman primate model for lentivirus infection and AIDS. To this end, we constructed a simian immunodeficiency virus/HIV-1 subtype-E (SHIV) recombinant clone by replacing the env ectodomain of the SHIV-33 clone with the env ectodomain from the subtype-E strain HIV-1(CAR402), which was isolated from an individual in the Central African Republic. Virus from this recombinant clone, designated SHIV-E-CAR, replicated efficiently in macaque peripheral blood mononuclear cells. Accordingly, juvenile macaques were inoculated with cell-free SHIV-E-CAR by the intravenous or intravaginal route; virus replicated in these animals but did not produce hematological abnormalities. In an attempt to elicit the pathogenic potential of the recombinant clone, we serially passaged this viral clone via transfusion of blood and bone marrow through juvenile macaques to produce SHIV-E-P4 (fourth-passage virus). The serially passaged virus established productive infection and CD4(+) T-cell depletion in juvenile macaques inoculated by either the intravenous or the intravaginal route. Determination of the coreceptor usage of SHIV-E-CAR and serially passaged SHIV-E-P4 indicated that both of these viruses utilized CXCR4 as a coreceptor. In summary, the serially passaged SHIV subtype-E chimeric virus will be important for studies aimed at developing a nonhuman primate model for analyzing the functions of subtype-E env genes in viral transmission and pathogenesis and for vaccine challenge experiments with macaques immunized with HIV-1 env antigens.

Protective immunity against feline immunodeficiency virus induced by inoculation with vif-deleted proviral DNA.

To determine whether live-attenuated feline immunodeficiency virus (FIV) proviral DNA will induce protective immunity, a plasmid clone constructed with a FIV provirus containing a deletion in the viral accessory gene vif (FIV-pPPR-Deltavif) was inoculated as proviral DNA into four cats by the intramuscular route. After 43 weeks, these cats were boosted with the same proviral plasmid. Analysis of peripheral blood mononuclear cells at several time points after the primary and booster inoculations revealed no detectable virus or proviral DNA. At 6 weeks after the booster, immunized cats and additional naive control cats were challenged with a cell-free preparation of the infectious biological isolate FIV-PPR by the intraperitoneal route. Virus was detected after challenge in unvaccinated control cats but not in any of the FIV-pPPR-Deltavif-immunized cats. Both FIV Gag- and Env-specific cytotoxic T lymphocyte (CTL) activities were detected in peripheral blood cells of control cats after challenge infection, whereas only one of four cats immunized with FIV-pPPR-Deltavif DNA exhibited a measurable CTL response to Env following challenge. Although anti-Gag antibodies were not detected after both proviral DNA inoculation and challenge, anti-Env antibodies were found in FIV-pPPR-Deltavif-immunized cats after vaccination as well as after challenge. These findings indicate that inoculation with FIV-pPPR-Deltavif proviral DNA induced resistance to challenge with infectious FIV and that a vif deletion mutant may provide a relatively safe attenuated lentiviral vaccine.

The intracytoplasmic domain of the Env transmembrane protein is a locus for attenuation of simian immunodeficiency virus SIVmac in rhesus macaques.

The human and simian immunodeficiency virus (HIV-1 and SIVmac) transmembrane proteins contain unusually long intracytoplasmic domains (ICD-TM). These domains are suggested to play a role in envelope fusogenicity, interaction with the viral matrix protein during assembly, viral infectivity, binding of intracellular calmodulin, disruption of membranes, and induction of apoptosis. Here we describe a novel mutant virus, SIVmac-M4, containing multiple mutations in the coding region for the ICD-TM of pathogenic molecular clone SIVmac239. Parental SIVmac239-Nef+ produces high-level persistent viremia and simian AIDS in both juvenile and newborn rhesus macaques. The ICD-TM region of SIVmac-M4 contains three stop codons, a +1 frameshift, and mutation of three highly conserved, charged residues in the conserved C-terminal alpha-helix referred to as lentivirus lytic peptide 1 (LLP-1). Overlapping reading frames for tat, rev, and nef are not affected by these changes. In this study, four juvenile macaques received SIVmac-M4 by intravenous injection. Plasma viremia, as measured by branched-DNA (bDNA) assay, reached a peak at 2 weeks postinoculation but dropped to below detectable levels by 12 weeks. At over 1.5 years postinoculation, all four juvenile macaques remain healthy and asymptomatic. In a subsequent experiment, four neonatal rhesus macaques were given SIVmac-M4 intravenously. These animals exhibited high levels of viremia in the acute phase (2 weeks postinoculation) but are showing a relatively low viral load in the chronic phase of infection, with no clinical signs of disease for 1 year. These findings demonstrated that the intracytoplasmic domain of the transmembrane Env (Env-TM) is a locus for attenuation in rhesus macaques.

Mucosal transmission of pathogenic CXCR4-utilizing SHIVSF33A variants in rhesus macaques.

Infection of macaques with chimeric simian/human immunodeficiency virus (SHIV) expressing the envelope protein of HIV-1 provides a model system for studying HIV-1 infection in humans. To this end, four rhesus macaques (Macaca mulatta) were given a single intravaginal (IVAG) inoculation of cell-free SHIVSF33A and longitudinal samples of peripheral blood and lymph nodes were analyzed for viremia, antigenemia, and various T-cell populations. Rhesus macaques infected IVAG with SHIVSF33A demonstrated a dramatic decrease in the CD4(+) PBMC subset in the initial weeks after viral exposure, a time that corresponded to peak in plasma viremia and antigenemia. Within 4 months of SHIVSF33A inoculation, partial to complete rebound of the CD4(+) PBMC was seen in these animals. Notably, the regeneration of the CD4(+) subset was associated with regeneration of the naive T-cell population and was concordant with clearance of plasma viremia. DNA heteroduplex tracking assays revealed transmission of minor variants within the SHIVSF33A inoculum to the IVAG-inoculated animals. The cell-free SHIVSF33A inoculum as well as virus isolated from animals early after transmission used the chemokine molecule CXCR4 as the primary cellular coreceptor, demonstrating that viruses expressing envelope glycoproteins of the syncytia inducing (SI) phenotype can be transported across the vaginal mucosa. Although none of the animals has yet to develop clinical symptoms of simian AIDS (SAIDS), infectious virus and viral nucleic acids could be persistently isolated from each animal. Furthermore, animals transfused with blood from IVAG-infected macaques drawn 2 weeks after inoculation suffered a more profound and sustained CD4(+) T-cell loss, persistent plasma viremia, and the development of SAIDS in one animal, indicating that IVAG-passaged SHIVSF33A was pathogenic. Taken together, these results establish that a pathogenic CXCR4-utilizing SHIVSF33A species crossed the cervicovaginal mucosa. Different courses of infection in the IVAG versus transfusion animals suggest that host-mediated responses elicited upon transmission across mucosal barriers may serve to limit viral replication and delay disease progression in the IVAG-infected animals.

Infection of baboons with a simian immunodeficiency virus/HIV-1 chimeric virus constructed with an HIV-1 Thai subtype E envelope.

OBJECTIVE: To construct an infectious chimeric simian immunodeficiency virus/HIV-1 (SHIV) with the envelope of a Thai subtype E HIV-1 strain for use in a non-human primate model. METHODS: A novel SHIV genome was derived using the sequences of the ectodomain of the envelope gene from the Thai subtype E strain, HIV-1(9466). This SHIV (SHIV9466.33) was recovered by cocultivation from human peripheral blood mononuclear cells (PBMC) after transfection of human rhabdosarcoma cells. Rhesus macaque and baboon PBMC were screened in vitro for susceptibility to infection with SHIV9466.33. After successful infection of baboon PBMC, four animals were inoculated intravenously with SHIV9466.33 and monitored for plasma viral RNA, virus isolation from the PBMC, seroconversion, T-cell subsets, and signs of disease. RESULTS: SHIV9466.33 was able to infect PBMC from 12 out of 14 baboons. All four of the baboons selected for in vivo inoculation became infected. Peak plasma viral RNA levels of 8000 to 700,000 RNA copies/ml were measured at 2 weeks post-inoculation. Virus was isolated from the PBMC of all four baboons during acute infection, and all seroconverted. Although transient declines in CD4+ T-cells were observed during early infection, CD4+ levels remained within normal ranges thereafter. In contrast, in vitro cultures of PBMC of four rhesus macaques were not susceptible to infection with SHIV9466.33. CONCLUSION: SHIV9466.33 containing an HIV-1 subtype E envelope displayed tropism for baboon PBMC but not for rhesus macaque PBMC. This chimeric virus established infection and induced antiviral antibodies in baboons inoculated by the intravenous route with cell-free virus. Thus, infection of baboons with SHIV9466.33 will serve as an important animal model for future studies of HIV-1 vaccine efficacy.

Neuropathogenesis induced by rhesus cytomegalovirus in fetal rhesus monkeys (Macaca mulatta).

Rhesus cytomegalovirus (RhCMV) infection of rhesus macaques offers opportunities to analyze mechanisms of CMV pathogenesis in a primate species. Four fetal rhesus monkeys were inoculated intraperitoneally with RhCMV early in the second trimester, and pregnancies were terminated by hysterotomy during the third trimester. Three fetuses had evidence of severe CMV disease, including intrauterine growth restriction, ventriculomegaly, microcephaly, lissencephaly, and extensive degenerative changes of the cerebral parenchyma. Histopathologic examination revealed polymicrogyria, gliosis, leptomeningitis, periventricular calcifications, and inclusion-bearing cells. These results demonstrate that the developing macaque brain is susceptible to infection with RhCMV early in the second trimester and that intrauterine infection results in neuropathologic outcomes similar to those observed in humans congenitally infected with CMV.

Importance of the intracytoplasmic domain of the simian immunodeficiency virus (SIV) envelope glycoprotein for pathogenesis.

SIVmac1A11 and SIVmac239 are nonpathogenic and pathogenic molecular clones in rhesus macaques, respectively. Although these viruses exhibit approximately 98% nucleotide and amino acid sequence homology, differences are found in the length of the translation frames for several genes. SIVmac239 has a premature stop codon in nef, whereas SIVmac1A11 has a premature stop codon in vpr and two premature stop codons in the intracytoplasmic domain of the env-transmembrane (TM) subunit. Recombinant viruses, constructed through reciprocal exchange of large DNA restriction enzyme fragments between SIVmac1A11 and SIVmac239, were evaluated in adult rhesus macaques. This in vivo analysis revealed that two or more regions of the SIVmac genome were essential for high virus load and disease progression (Marthas et al., 1993. J. Virol. 67, 6047-6055). An important gap in knowledge remaining from this study was whether the premature stop codons in env-TM of recombinant virus SIV1A11/239gag-env/1A11 (Full-length vpr and nef, two stop codons in env-TM) reverted to coding triplets in vivo. Here, we report that viral sequences in macaques, which succumbed to an AIDS-like disease after infection with SIV1A11/239gag-env/1A11, exhibited reversion of both env-TM stop codons. In addition, antibodies to the intracytoplasmic domain of env-TM were detected in macaques containing revertant virus and showing disease; this finding indicates that this domain of the env glycoprotein was expressed in vivo. Thus selection for viral variants with full-length env-TM demonstrated that the cytoplasmic domain of the SIVmac env glycoprotein plays a role in viral persistence and immunodeficiency in primates.

Cloning and characterization of rhesus cytomegalovirus glycoprotein B.

Rhesus cytomegalovirus (RhCMV) infection of rhesus macaques is an important model to investigate critical issues of cytomegalovirus biology. To better understand host immunological responses to viral glycoproteins, the glycoprotein B (gB) gene of RhCMV was molecularly cloned, sequenced and characterized. Transcription analysis revealed that RhCMV gB was transcribed as a late gene. The RhCMV gB gene encoded a predicted protein of 854 amino acids that was 60% identical/75% similar to the human CMV (HCMV) gB protein. The region of HCMV gB proposed to be responsible for virus binding to host cells, fusion and cell-to-cell spread was the most highly conserved region with RhCMV gB (74% identity/85% similarity). Conserved elements included 11 of 12 cysteine residues, 14 of 16 potential N-linked glycosylation sites and cross-reactive epitopes. Metabolic labelling experiments demonstrated that RhCMV gB was proteolytically processed similarly to HCMV gB. These results are critical for investigating virus-host relationships in CMV-infected primates.

Transgenic dissection of HIV genes involved in lymphoid depletion.

Transgenic mice carrying an HIV provirus, with selective deletion of all three structural genes, developed extensive lymphoid depletion which was detected not only in the spleen and lymph nodes but also in the thymus. Mice with a high level of HIV gene expression developed acute disease which resulted in premature death, and mice with a low level of viral transcripts developed chronic disease with long-term survival. Neither HIV replication nor the envelope glycoprotein (gp120) was required for T cell depletion. Despite abundant viral gene expression early in life, cell death did not become evident until about the time of full lymphoid maturation, suggesting that thymopoiesis was not affected. The more mature T cells in the peripheral lymphoid organs and in the thymic medulla were less sensitive to the apoptotic process than the immature T cells in the thymic cortex. Gradual depletion of the T cell compartment in the peripheral lymphoid organs was intimately accompanied by the reciprocal expansion of the B cell compartment, resulting in the almost complete replacement of T lymphocytes with B immunoblasts in lymph nodes. Unlike T cells, which showed abundant HIV gene expression, B cells did not. The transgenic approach may help identify the HIV nonstructural gene(s) responsible for immune deficiency and help facilitate dissection of its role in inducing apoptosis.

Activation of PAK by HIV and SIV Nef: importance for AIDS in rhesus macaques.

BACKGROUND: The primate lentiviruses, human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV), encode a conserved accessory gene product, Nef. In vivo, Nef is important for the maintenance of high virus loads and progression to AIDS in SIV-infected adult rhesus macaques. In tissue culture cells expressing Nef, this viral protein interacts with a cellular serine kinase, designated Nef-associated kinase. RESULTS: This study identifies the Nef-associated kinase as a member of the p21-activated kinase (PAK) family of kinases and investigates the role of this Nef-associated kinase in vivo. Mutants of Nef that do not associate with the cellular kinase are unable to activate the PAK-related kinase in infected cells. To determine the role of cellular kinase association in viral pathogenesis, macaques were infected with SIV containing point-mutations in Nef that block PAK activation. Virus recovered at early time points after inoculation with mutant virus was found to have reverted to prototype Nef function and sequence. Reversion of the kinase-negative mutant to a kinase-positive genotype in macaques infected with the mutant virus preceded the induction of high virus loads and disease progression. CONCLUSIONS: Nef associates with and activates a PAK-related kinase in lymphocytes infected in vitro. Moreover, the Nef-mediated activation of a PAK-related kinase correlates with the induction of high virus loads and the development of AIDS in the infected host. These findings reveal that there is a strong selective pressure in vivo for the interaction between Nef and the PAK-related kinase.

The simian foamy virus type 1 transcriptional transactivator (Tas) binds and activates an enhancer element in the gag gene.

Simian and human foamy viruses (SFV and HFV) encode a transcriptional transactivator, Tas, which governs the levels of viral transcripts initiated by both the promoter in the long terminal repeat (LTR) and the internal promoter (IP) located within the env gene of these viruses. Tas-responsive target elements,(TRE) LTR in the LTR and (TRE) IP in the env gene, are located 5' of the TATA box in both viral promoters and function as orientation- and position-independent enhancers. We have identified a strong Tas-responsive element, designated TRE (GP), near the 3' end of the gag gene and preceding the pol gene of SFV-1. In transient-expression assays with plasmids containing reporter genes, a 59-bp DNA fragment containing TRE (GP) (nucleotides 2224 to 2282) functioned as an enhancer element, dependent on Tas, in several cell types and in the context of a heterologous basal promoter. DNase footprinting revealed that the fusion protein glutathione S-transferase-Tas, purified from genetically engineered bacteria, interacts with about 40 hp (nucleotides 2237 to 2279) in the TRE (GP). A low degree of sequence homology was noted between TRE (GP) and TRE (IP). In virus-infected cells, novel transcripts with 5' ends immediately upstream from the reverse transcriptase translation frame (nucleotides 2611 to 5778) were identified. Upstream of the start site for these transcripts is a TATA box (nucleotides 2575 to 2579), which was required for transcription in transient-expression assays. Although a spliced mRNA initiated in the viral LTR is implicated in the synthesis of the HFV Pol polyprotein which encodes protease, reverse transcriptase, and integrase, it is possible that SFV-1 contains a promoter within the pol gene for initiating a reverse transcriptase transcript. Taken together, these studies define a novel Tas-responsive enhancer element, which binds the viral transactivator, and a potential promoter within the pol gene.

Nucleotide sequence and molecular analysis of the rhesus cytomegalovirus immediate-early gene and the UL121-117 open reading frames.

Cytomegalovirus (CMV) has been isolated from many nonhuman primates, including rhesus macaques (Macaca mulatta). To better understand the molecular biology of rhesus CMV (RhCMV), a 9.2-kb DNA restriction fragment spanning the immediate-early (IE) gene has been molecularly cloned and sequenced. Open reading frames (ORF) have been identified and transcripts mapped for regions corresponding to exons 1, 2, 3, and 4 of the IE1 protein of human CMV (HCMV) and to exons 1, 2, 3, and 5 of IE2. The predicted RhCMV IE1 protein was 29 and 40% identical with the HCMV and African green monkey (AGM) CMV IE1 proteins, respectively, and the predicted RhCMV IE2 protein was 48 and 65% identical with the HCMV and AGM CMV IE2 proteins, respectively. Five additional ORF 3' to the RhCMV IE gene were identified which contained significant homologies with the HCMV UL121-UL117 ORF. The predicted translation products ranged from 29 to 47% identical with, and 52 to 66% similarity to the corresponding ORF of HCMV. Conservation of nucleic and amino acid sequences, and colinearity of genes, between primate CMV genomes contribute to a better understanding of primate CMV evolution, regulation, and pathogenesis.

Persistent infection of rhesus macaques with T-cell-line-tropic and macrophage-tropic clones of simian/human immunodeficiency viruses (SHIV).

To elucidate the functions of human immunodeficiency virus type 1 (HIV-1) genes in a nonhuman primate model, we have constructed infectious recombinant viruses (chimeras) between the pathogenic molecular clone of simian immunodeficiency virus (SIV) SIVmac239 and molecular clones of HIV-1 that differ in phenotypic properties controlled by the env gene. HIV-1SF33 is a T-cell-line-tropic virus which induces syncytia, and HIV-1SF162 is a macrophage-tropic virus that does not induce syncytia. A DNA fragment encoding tat, rev, and env (gp160) of SIVmac239 has been replaced with the counterpart genetic region of HIV-1SF33 and HIV-1SF162 to derive chimeric recombinant simian/human immunodeficiency virus (SHIV) strains SHIVSF33 and SHIVSF162, respectively. In the acute infection stage, macaques inoculated with SHIVSF33 had levels of viremia similar to macaques infected with SIVmac239, whereas virus loads were 1/10th to 1/100th those in macaques infected with SHIVSF162. Of note is the relatively small amount of virus detected in lymph nodes of SHIVSF162-infected macaques. In the chronic infection stage, macaques infected with SHIVSF33 also showed higher virus loads than macaques infected with SHIVSF162. Virus persists for over 1 year, as demonstrated by PCR for amplification of viral DNA in all animals and by virus isolation in some animals. Antiviral antibodies, including antibodies to the HIV-1 env glycoprotein (gp160), were detected; titers of antiviral antibodies were higher in macaques infected with SHIVSF33 than in macaques infected with SHIVSF162. Although virus has persisted for over 1 year after inoculation, these animals have remained healthy with no signs of immunodeficiency. These findings demonstrate the utility of the SHIV/macaque model for analyzing HIV-1 env gene functions and for evaluating vaccines based on HIV-1 env antigens.

Characterization of the internal promoter of simian foamy viruses.

Simian and human foamy viruses (HFV and SFV), genetically related members of the spumavirus genus of retroviruses, have complex genome structures which encode the gag, pol, and env genes for virion proteins as well as additional open reading frames. One of these open reading frames is a viral transactivator, encoded by genes designated taf for SFV and bel-1 for HFV, which augments transcription directed by the long terminal repeat (LTR) through cis-acting targets in the U3 domain of the LTR. Recently, an internal transcriptional promoter has been identified in sequences within the 3' end of the HFV env gene (M. Lochelt, W. Muranyi, and R. M. Flugel, Proc. Natl. Acad. Sci. 90:7317-7321, 1993). We have demonstrated by using transient expression assays in several tissue culture cell lines and by analyzing viral transcripts in infected cells that SFV-1 from a rhesus macaque and SFV-3 from an African green monkey also encode an internal promoter in the env gene. Transcription directed by the internal promoters of SFV-1 and SFV-3 is activated by the taf-1 and taf-3 gene products, respectively, in several cell types. The importance of a TATA box for the SFV-1 internal promoter was established by site-specific mutagenesis, and the 5' ends of transcripts initiating in the internal promoter have been determined. cis-acting sequences in the SFV-1 env gene required for the response to taf-1 are contained within a 121-bp element located 5' to the TATA box in the internal promoter. This taf-1-responsive element in the internal promoter functions in a position- and orientation-independent fashion in a heterologous promoter and thus has the properties of an enhancer which depends on taf-1 activity. Alignments reveal that the SFV-1 internal promoter and the SFV-1 LTR have little sequence relatedness. Cross-transactivation studies show that the transactivators of SFV-1 and HFV function on the internal promoter and LTR of the homologous virus but not on the heterologous virus. In summary, the genomes of simian and human foamy viruses direct viral transcription through both the promoter in the LTR and an internal promoter within the env gene, and each promoter contains unique enhancer-like elements regulated by the viral transactivator.