The prophylactic and therapeutic properties of 3'-azido-3'-deoxythymidine (AZT) against simian immunodeficiency virus (SIV) infection were tested in four 3-month-old rhesus macaques. The infant monkeys were inoculated intravenously with a low dose (1 to 10 100%v animal infectious doses) of uncloned SIVmac* The monkeys were treated orally with 50 mg of AZT per kg of body weight every 8 h; two animals were started on treatment 2 h prior to virus inoculation, and two animals were started on treatment 6 weeks later. All four animals were treated for a period of 6 to 10 weeks. Outward signs of AZT toxidcity were absent, but a mild macrocytic anemia occurred soon after therapy was started and resolved shortly after it was discontinued. The two infants that were begun on AZT treatment 2 h prior to virus inoculation never became infected, as demonstrated by the inability to detect cell-free or cell-associated virus in the blood, proviral DNA in peripheral blood mononuclear cells, or anti-SIV antibodies. AZT administration over a 10-week period had no detectable effect on the course of disease in the two animals that were begun on treatment after the infection had been established. In addition to demonstrating the prophylactic effect of AZT against low-dose SIV exposure, the study demonstrated the ease with which infant rhesus macaques can be used for antiretroviral drug testing.

SIV/HIV-1 (SHIV) chimeric clones, constructed by substituting portions of the pathogenic molecular clone SIVmac239 with counterpart portions from HIV-1 clones, provide a means to analyze functions of selected HIV-1 genes in vivo in nonhuman primates. Our studies focused on SHIVSF33, which contains the vpu, tat, rev, and env genes of the cytopathic, T-cell line tropic clone HIV-1sf33 (subtype-B); this clone has a premature stop codon in the vpu gene. In three juvenile macaques inoculated intravenously with SHIVSF33, low-level persistent infection was established; no disease was observed for a period of >2 years. However, at approximately 16 months p.i., one of four SHIVSF33-infected juvenile macaques exhibited an increase in virus load, depletion of CD4(+) T cells in peripheral blood and lymph nodes, and other symptoms of simian AIDS (SAIDS). Virus recovered from this animal in the symptomatic stage was designated SHIVSF33a (A, adapted); this virus displayed multiple amino acid sequence changes throughout the HIV-1 env gene compared with the input SHIVSF33 clone. Additionally, a mutation in all clones from SHIVSF33a restored the open reading frame for the vpu gene. In vitro evaluations in tissue-culture systems revealed that SHIVSF33a replicated to higher levels and exhibited greater cytopathicity than SHIVSF33. Furthermore cloned env genes for SHIVSF33a were more fusogenic in a cell-fusion assay compared with the env gene of the SHIVSF33. Intravenous inoculation of SHIVsf33a into juvenile and newborn macaques resulted in a rapid decline in CD4(+) T cells to very low levels and development of a fatal AIDS-like disease. A cell-free preparation of this pathogenic chimeric virus also established persistent infection when applied to oral mucosal membranes of juvenile macaques and produced a fatal AIDS-like disease. These studies on pathogenic SHIVSF33a establish the basis for further investigations on the role of the HIV-1 env gene in virus adaptation and in mechanism(s) of immunodeficiency in primates; moreover, the chimeric virus SHIVSF33a can play a role in elucidating mucosal membrane transmission and development of antiviral vaccines in newborns as well as juvenile and adult macaques.

Human cytomegalovirus (HCMV) establishes and maintains a lifelong persistence following infection in an immunocompetent host. The determinants of a stable virus-host relationship are poorly defined. A nonhuman primate model for HCMV was used to investigate virological and host parameters of infection in a healthy host. Juvenile rhesus macaques (Macaca mulatta) were inoculated with rhesus cytomegalovirus (RhCMV), either orally or intravenously (i.v.), and longitudinally necropsied. None of the animals displayed clinical signs of disease, although hematologic abnormalities were observed intermittently in i.v. inoculated animals. RhCMV DNA was detected transiently in the plasma of all animals at 1 to 2 weeks postinfection (wpi) and in multiple tissues beginning at 2 to 4 wpi. Splenic tissue was the only organ positive for RhCMV DNA in all animals. The location of splenic cells expressing RhCMV immediate-early protein 1 (IE1) in i.v. inoculated animals changed following inoculation. At 4 to 5 wpi, most IE1-positive cells were perifollicular, and at 25 wpi, the majority were located within the red pulp. All animals developed anti-RhCMV immunoglobulin M (IgM) antibodies within 1 to 2 wpi and IgG antibodies within 2 to 4 wpi against a limited number of viral proteins. Host reactivity to RhCMV proteins increased in titer (total and neutralizing) and avidity with time. These results demonstrate that while antiviral immune responses were able to protect from disease, they were insufficient to eliminate reservoirs of persistent viral gene expression.

The simian immunodeficiency virus (SIV) nef gene is an important determinant of viral load and acquired immunodeficiency syndrome (AIDS) in macaques. A role(s) for the HIV-1 nef gene in infection and pathogenesis was investigated by constructing recombinant viruses in which the nef gene of the pathogenic molecular clone SIVmac239 nef was replaced with either HIV-1sf2nef or HIV-1sf33nef. These chimeras, designated SHIV-2nef and SHIV-33nef, expressed HIV-1 Nef protein and replicated efficiently in cultures of rhesus macaque lymphoid cells. In two SHIV-2nef-infected juvenile rhesus macaques and in one of two SHIV-33nef-infected juvenile macaques, virus loads remained at low levels in both peripheral blood and lymph nodes in acute and chronic phases of infection (for >83 weeks). In striking contrast, the second SHIV-33nef-infected macaque showed high virus loads during the chronic stage of infection (after 24 weeks). CD4+ T-cell numbers declined dramatically in this latter animal, which developed simian AIDS (SAIDS) at 47-53 weeks after inoculation; virus was recovered at necropsy at 53 weeks and designated SHIV-33Anef. Sequence analysis of the HIV-1sf33 nef gene in SHIV-33Anef revealed four consistent amino acid changes acquired during passage in vivo. Interestingly, one of these consensus mutations generated a tyr-x-x-leu (Y-X-X-L) motif in the HIV-1sf33 Nef protein. This motif is characteristic of certain endocytic targeting sequences and also resembles a src-homology region-2 (SH-2) motif found in many cellular signaling proteins. Four additional macaques infected with SHIV-33Anef contained high virus loads, and three of these animals progressed to fatal SAIDS. Several of the consensus amino acid changes in Nef, including Y-X-X-L motif, were retained in these recipient animals exhibiting high virus load and disease. In summary, these findings indicate that the SHIV-33Anef chimera is pathogenic in rhesus macaques and that this approach, i.e., construction of chimeric viruses, will be important for analyzing the function(s) of HIV-1 nef genes in immunodeficiency in vivo, testing antiviral therapies aimed at inhibiting AIDS, and investigating adaptation of this HIV-1 accessory gene to the macaque host.

The nef gene of the human and simian immunodeficiency viruses (HIV and SIV) is dispensable for viral replication in T-cell lines; however, it is essential for high virus loads and progression to simian AIDS (SAIDS) in SIV-infected adult rhesus macaques. Nef proteins from HIV type 1 (HIV-1), HIV-2, and SIV contain a proline-Xaa-Xaa-proline (PxxP) motif. The region of Nef with this motif is similar to the Src homology region 3 (SH3) ligand domain found in many cell signaling proteins. In virus-infected lymphoid cells, Nef interacts with a cellular serine/threonine kinase, designated Nef-associated kinase (NAK). In this study, analysis of viral clones containing point mutations in the nef gene of the pathogenic clone SIVmac239 revealed that several strictly conserved residues in the PxxP region were essential for Nef-NAK interaction. The results of this analysis of Nef mutations in in vitro kinase assays indicated that the PxxP region in SIV Nef was strikingly similar to the consensus sequence for SH3 ligand domains possessing the minus orientation. To test the significance of the PxxP motif of Nef for viral pathogenesis, each proline was mutated to an alanine to produce the viral clone SIVmac239-P104A/P107A. This clone, expressing Nef that does not associate with NAK, was inoculated into seven juvenile rhesus macaques. In vitro kinase assays were performed on virus recovered from each animal; the ability of Nef to associate with NAK was restored in five of these animals as early as 8 weeks after infection. Analysis of nef genes from these viruses revealed patterns of genotypic reversion in the mutated PxxP motif. These revertant genotypes, which included a second-site suppressor mutation, restored the ability of Nef to interact with NAK. Additionally, the proportion of revertant viruses increased progressively during the course of infection in these animals, and two of these animals developed fatal SAIDS. Taken together, these results demonstrated that in vivo selection for the ability of SIV Nef to associate with NAK was correlated with the induction of SAIDS. Accordingly, these studies implicate a role for the conserved SH3 ligand domain for Nef function in virally induced immunodeficiency.