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Attenuated molecular clones of simian immunodeficiency virus (SIVmac) are important tools for studying the correlates of protective immunity to lentivirus infection in nonhuman primates. The most highly attenuated SIVmac mutants fail to induce disease but also fail to induce immune responses capable of protecting macaques from challenge with pathogenic virus. We recently described a novel attenuated virus, SIVmac-M4, containing multiple mutations in the transmembrane protein (TM) intracytoplasmic domain. This domain has been implicated in viral assembly, infectivity, and cytopathogenicity. Whereas parental SIVmac239-Nef(+) induced persistent viremia and simian AIDS in rhesus macaques, SIVmac-M4 induced transient viremia in juvenile and neonatal macaques, with no disease for at least 1 year postinfection. In this vaccine study, 8 macaques that were infected as juveniles (n = 4) or neonates (n = 4) with SIVmac-M4 were challenged with pathogenic SIVmac251 administered through oral mucosa. At 1 year postchallenge, six of the eight macaques had low to undetectable plasma viremia levels. Assays of cell-mediated immune responses to SIVmac Gag, Pol, Env, and Nef revealed that all animals developed strong CD8(+) T-cell responses to Gag after challenge but not before. Unvaccinated control animals challenged with SIVmac251 developed persistent viremia, had significantly weaker SIV-specific T-cell responses, and developed AIDS-related symptoms. These findings demonstrate that SIVmac-M4, which contains a full-length Nef coding region and multiple point mutations in the TM, can provide substantial protection from mucosal challenge with pathogenic SIVmac251.

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.

Rhesus macaques infected with simian immunodeficiency virus (SIV) containing either a large nef deletion (SIVmac239Delta(152)nef) or interleukin-2 in place of nef developed high virus loads and progressed to simian AIDS. Viruses recovered from both juvenile and neonatal macaques with disease produced a novel truncated Nef protein, tNef. Viruses recovered from juvenile macaques infected with serially passaged virus expressing tNef exhibited a pathogenic phenotype. These findings demonstrated strong selective pressure to restore expression of a truncated Nef protein, and this reversion was linked to increased pathogenic potential in live attenuated SIV vaccines.

Simian foamy virus type 1 (SFV-1), a member of the Spumavirinae subfamily of retroviruses, encodes a transcriptional transactivator (taf) that strongly augments gene expression directed by the viral long terminal repeat (LTR) (A. Mergia, K. E. S. Shaw, E. Pratt-Lowe, P. A. Barry, and P. A. Luciw, J. Virol. 65:2903-2909, 1991). This report describes cis-acting regulatory elements in the LTR that control viral gene expression. A series of LTR mutants and hybrid promoter constructs have been analyzed in transient expression assays for responsiveness to Taf. The targets for transactivation have been mapped to two regions of the U3 domain of the LTR, between positions -1196 and -880 and between positions -403 and -125 (+1 represents the transcription initiation site). No significant nucleotide sequence homology between these two regions is noted; thus, the SFV-1 taf gene acts through at least two distinct sequence elements in the LTR. The target contained between positions -403 and -125 acts independently of orientation, in different cell types and species, and in the context of a heterologous promoter. Thus, the target element between positions -403 and -125 has properties of a transcriptional enhancer. The observation that two distinct elements in the SFV-1 LTR are targets for transcriptional transactivation is novel with respect to observations for other retroviral systems. The R-U5 region of the SFV-1 LTR down-regulates transactivation by severalfold. Computer analysis of the R-U5 region revealed a secondary structure with a free-energy level of -74 kcal (ca. -310,000 J); this structural feature may account for the inhibitory effect on gene expression directed by the LTR. Taf of SFV-1 had no effect on gene expression directed by the LTR of the related human foamy virus, whereas Taf transactivates gene expression directed by the LTRs of the human and simian immunodeficiency viruses. Comparative functional analysis of Taf on homologous and heterologous LTRs may facilitate elucidation of the mechanism of transactivation of foamy viruses.

Simian foamy virus type 1 (SFV-1), a member of spumavirus subfamily of retroviruses, encodes a transcriptional transactivator that functions to strongly augment gene expression directed by the viral long terminal repeat (LTR). The objective of this study was to identify the viral gene responsible for transactivation. Nucleotide sequences between the env gene and the LTR of SFV-1 were determined. The predicted amino acid sequence revealed two large open reading frames (ORFs), designated ORF-1 (311 amino acids) and ORF-2 (422 amino acids). In the corresponding region of the human foamy virus, three ORFs (bel-1, bel-2, and bel-3) have been identified (R. M. Flugel, A. Rethwilm, B. Maurer, and G. Darai, EMBO J. 6:2077-2084, 1987). Pairwise comparisons of the ORF-1 and ORF-2 with bel-1 and bel-2 show small clusters of homology; less than 39% overall homology of conserved amino acids is observed. A counterpart for human foamy virus bel-3 is not present in the SFV-1 sequence. Three species of viral RNA have been identified in cells infected with SFV-1; an 11.5-kb RNA representing full-length transcripts, a 6.5-kb RNA representing the env message, and a 2.8-kb RNA from the ORF region. Analysis of a cDNA clone encoding the ORF region of SFV-1 reveals that the 2.8-kb message is generated by complex splicing events involving the 3' end of the env gene. In transient expression assays in cell lines representing several species. ORF-1 was shown to be necessary and sufficient for transactivating viral gene expression directed by the SFV-1 LTR. The target for transactivation is located in the U3 domain of the LTR, upstream from position - 125 (+ 1 represents the transcription initiation site). We propose that OFF-1 of SFV-1 be designated the transcriptional transactivator of foamy virus (taf).

Simian foamy viruses, members of the spumavirus subfamily of retroviruses, are found in a variety of nonhuman primates and, as yet, remain to be characterized with respect to genetic structure and regulation of viral gene expression. The genome of simian foamy virus type 1 (SFV-1), an isolate from rhesus macaques, has been molecularly cloned, and the role of the viral long terminal repeat (LTR) in transcriptional control has been investigated. The SFV-1 LTR is 1,621 base pairs long, and sequence comparisons with human foamy virus revealed a pattern of clustered homology. A cap site in the LTR was identified by analysis of SFV-1 transcripts in infected cells. Transient expression assays in cell lines representing several species and different cell types showed that the SFV-1 LTR has low basal activity in uninfected cells, whereas LTR-directed expression is greatly increased in cells infected with SFV-1. This transactivation is mediated by a mechanism involving increases in steady-state levels of viral transcripts. Thus, the SFV-1 genome encodes a transactivator that functions on the LTR at the transcriptional level.

We have molecularly cloned and sequenced a portion of the simian foamy virus type 1 (SFV-1); open reading frames representing the endonuclease domain of the polymerase (pol) and the envelope (env) genes were identified by comparison with the human foamy virus (HFV). Unlike the HFV genomic organization, the SFV-1 pol gene overlaps the env gene; thus, the open reading frames reported for HFV between pol and env is not present in SFV-1. Comparisons of predicted amino acid sequences of HFV and SFV-1 reveal that the endonuclease domains of the pol genes are about 84% related. The region predicted to encode the SFV-1 extracellular env domain is 569 codons; SFV-1 and HFV have 64% amino acid similarity in this env domain. The predicted hydrophobic transmembrane env proteins of both HFV and SFV-1 show about 73% similarity. A total of 16 potential glycosylation sites are found in SFV-1 env, and 15 are found in HFV; 11 are shared. SFV-1 has 25 cysteine residues, and HFV has 23 residues; all 23 cysteine residues of HFV are conserved in SFV-1. This sequence analysis reveals that the human and simian foamy viruses are highly related.