Animal models for AIDS pathogenesis.
Explore the source record for details and available documents.
Acquired Immunodeficiency Syndrome↗
Biomedical subjects
H W Kestler
Publications and source records attributed to H W Kestler.
Growth properties of HSIVnef: HIV-1 containing the nef gene from pathogenic molecular clone SIVmac239.
To elucidate the function of nef, we constructed infectious chimeric clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) of macaques, by deleting a part of the nef sequence from the HIV-1 genome and inserting the entire nef gene from the pathogenic molecular clone, SIVmac239. We have named this construct HSIVnef. While there was no difference in the replication kinetics of HSIVnef+ and HSIVnef- in transformed cell lines, there was a profound difference in the replication of these virus in primary cells. These data indicated that the SIV nef gene could complement the functions of the HIV-1 nef gene in the context of the HIV-1 genome, providing further evidence that results observed in the macaque model are relevant to HIV infections in man. HSIVnef is a useful model for the study of the nef gene.
Animals↗
A live-virus "suicide" vaccine for human immunodeficiency virus.
A vaccine that uses a live, attenuated human immunodeficiency virus (HIV) may offer the best hope of a vaccine against acquired immunodeficiency syndrome (AIDS). A recent improvement should increase the safety of the live-virus approach: a "suicide gene" inserted into the viral RNA, which causes infected cells to die when treated with ganciclovir. We envision using this strategy not only to prevent AIDS, but also to treat it.
AIDS Vaccines↗
A candidate live inactivatable attenuated vaccine for AIDS.
The recent discovery of long term AIDS nonprogressors who harbor nef-attenuated HIV suggests that a naturally occurring live vaccine for AIDS may already exist. Animal models have shown that a live vaccine for AIDS, attenuated in nef, is the best candidate vaccine. There are considerable risks, real and perceived, with the use of live HIV vaccines. We have introduced a conditional lethal genetic element into HIV-1 and simian immunodeficiency virus (SIV) molecular clones deleted in nef. The antiviral strategy we employed targets both virus replication and the survival of the infected cell. The suicide gene, herpes simplex virus thymidine kinase (tk), was expressed and maintained in HIV over long periods of time. Herpes simplex virus tk confers sensitivity to the antiviral activity of acyclic nucleosides such as ganciclovir (GCV). HIV-tk and SIV-tk replication were sensitive to GCV at subtoxic concentrations, and virus-infected cells were eliminated from tumor cell lines as well as primary cell cultures. We found the HIV-tk virus to be remarkably stable even after being cultured in media containing a low concentration of GCV and then challenged with the higher dose and that while GCV resistant escape mutants did arise, a significant fraction of the virus remained sensitive to GCV.
AIDS Vaccines↗
The nef gene from a long-term HIV type 1 nonprogressor.
We examined the nef gene of HIV-1 in a long-term nonprogressor to look for evidence suggesting an attenuated virus. The nef gene was previously shown to be required for induction of AIDS. Simian immunodeficiency virus (SIV) deleted in nef, while infectious, fails to sustain the high viral loads necessary for the induction of AIDS in infected adult rhesus monkeys. The human subject of this report was found to harbor virus (HIV-1 Sur25) encoding open-nef reading frames. However, the nef genes of this subject bore a signature point mutation: a cysteine at amino acid 138. The sequence at this position was identical in all clones examined over a 3-year period. When this sequence was compared to the sequence database for AIDS and human retroviruses at Los Alamos, New Mexico, several isolates from other asymptomatic individuals were also found to encode nef genes with a cysteine at position 138. Furthermore, Cys-138 was found in chimpanzee immunodeficiency virus (CIV), a lentivirus that is similar to HIV but does not cause AIDS in chimpanzees. Multiple cysteines are also found in the nef gene of African green monkey virus, SVIagm, including cysteine at the position analogous to Cys-138. While seroprevalence of SIVagm is high in the wild, there is no known disease associated with this virus. The pathogenic virus isolated from Asian macaques, SIVmac, encodes a Nef protein that has few cysteines. Although the virus HIVSur25 encodes a completely open-nef gene, the virus from this individual is similar to attenuated SIVmac (SIVmac239/nef-deletion) as well as HIV deleted in nef in its growth properties in H9 cells. Nef containing a cysteine at position 138 was shown to be responsible for determining the ability to grow in H9.
Amino Acid Sequence↗
Attenuated retrovirus vaccines and AIDS.
Explore the source record for details and available documents.
AIDS Vaccines↗
Upstream U3 sequences in simian immunodeficiency virus are selectively deleted in vivo in the absence of an intact nef gene.
Major transcriptional control elements are located within the U3 region of the long terminal repeats (LTRs) of lentivirus and other retroviral genomes. The nef auxiliary gene of simian immunodeficiency virus (SIV) and human immunodeficiency virus overlaps about 70% of the 450- to 560-bp-long U3 region present in these primate lentiviruses. We analyzed viral DNA sequences present in rhesus monkeys infected with a mutant of SIVmac containing a 182-bp deletion in the region of nef that does not overlap the LTR. Between 50 and 100% of the viral DNA molecules in eight of nine monkeys infected for 16 or more months contained additional deletions of 111 to 302 bp within the 517-bp U3 region. These deletions were contained within a 334-bp region of U3 that is overlapped by the nef reading frame, and they did not affect the polypurine tract, the NF-kappa B binding site, or other sequence elements in this same region that are important for transcription and replication. Such deletions were not detected in any of 41 PCR amplifications from 8 rhesus monkeys infected with wild-type SIV for 8 to 26 months, nor were they detected in 10 animals infected with vpr, vpx or vpr-vpx deletion mutants. These results indicate that, in the absence of an intact nef gene, these upstream U3 sequences are not advantageous for the virus.
Animals↗
Importance of the nef gene for maintenance of high virus loads and for development of AIDS.
When rhesus monkeys were infected with a form of cloned SIVmac239 having a premature stop signal at the 93rd codon of nef, revertants with a coding codon at this position quickly and universally came to predominate in the infected animals. This suggests that there are strong selective forces for open functional forms of nef in vivo. Although deletion of nef sequences had no detectable effect on virus replication in cultured cells, deletion of nef sequences dramatically altered the properties of virus in infected rhesus monkeys. Our results indicate that nef is required for maintaining high virus loads during the course of persistent infection in vivo and for full pathologic potential. Thus, nef should become a target for antiviral drug development. Furthermore, the properties of virus with a deletion in nef suggest a means for making live-attenuated strains of virus for experimental vaccine testing.
Animals↗
Nef genes of SIV.
Molecular clones of SIVmac were constructed that differed only in sequences within the nef gene. DEAE-transfection of viral DNA containing an open from of nef yielded virus that replicated with similar kinetics and to a similar extent in macaque peripheral blood lymphocyte (PBL) cultures as virus with a deletion or stop codon within nef. Rhesus monkeys that received each kind of molecularly cloned virus became infected. Our results additionally suggest that mutant forms of virus are selected in vitro while open, functional forms are selected in vivo. In animals infected with virus containing a stop codon within nef, reversion of the stop codon to a coding codon was demonstrated in five of five clones analyzed. These results indicate that nef is playing some role crucial to the virus life cycle in vivo.
Animals↗
Molecular changes associated with replication of simian immunodeficiency virus in human cells.
The SIVmac239 infectious clone does not have a premature stop codon in its transmembrane protein (TMP) region and it produces full-length (41 kilodalton, kDa) TMP in macaque peripheral blood lymphocytes (PBL) in vitro and in vivo. However, viruses with truncated forms of TMP (28kDa) are selected during propagation in human cell types; truncated forms arise from point mutations, CAG (glutamine) to TAG (stop), in the viral genome. These results document molecular changes associated with adaptation of SIVmac for growth in human cells.
Animals↗
Nonhuman primate lentiviruses: models for human infection.
Explore the source record for details and available documents.
Acquired Immunodeficiency Syndrome↗
Significance of premature stop codons in env of simian immunodeficiency virus.
The location of the translational termination codon for the transmembrane protein (TMP) varies in three infectious molecular clones of simian immunodeficiency virus from macaques (SIVmac). The SIVmac251 and SIVmac142 infectious clones have premature stop signals that differ in location by one codon; transfection of these DNAs into human HUT-78 cells yielded virus with a truncated TMP (28 to 30 kilodaltons [kDa]). The SIVmac239 infectious clone does not have a premature stop codon in its TMP-coding region. Transfection of HUT-78 cells with this clone initially yielded virus with a full-length TMP (41 kDa). At 20 to 30 days posttransfection, SIVmac239 virus with a 41-kDa TMP gradually disappeared coincident with the emergence of a virus with a 28-kDa TMP. Virus production dramatically increased in parallel with the emergence of a virus with a 28-kDa TMP. Sequence analysis of viral DNAs from these cultures showed that premature stop codons arising by point mutation were responsible for the change in size of the TMP with time. A similar selective pressure for truncated forms of TMP was observed when the SIVmac239 clone was transfected into human peripheral blood lymphocytes (PBL). In contrast, no such selective pressure was observed in macaque PBL. When the SIVmac239 clone was transfected into macaque PBL and the resultant virus was serially passaged in macaque PBL, the virus replicated very well and maintained a 41-kDa TMP for 80 days in culture. Macaque monkeys were infected with SIVmac239 having a 28-kDa TMP; virus subsequently recovered from T4-enriched lymphocytes of peripheral blood showed only the 41-kDa form of TMP. These results indicate that the natural form of TMP in SIVmac is the full-length 41-kDa TMP, just as in human immunodeficiency virus type 1. Viruses with truncated forms of TMP appear to result from mutation and selection during propagation in unnatural human cells.
Amino Acid Sequence↗
Use of infectious molecular clones of simian immunodeficiency virus for pathogenesis studies.
Three infectious molecular clones of SIVmac and one of HIV-2 exhibit remarkable variation in their biological properties despite similarities in genome organization and sequence relatedness. Cloned viruses differed in their ability to grow in various cultured cells, in their ability to infect macaques, and in the location of the env stop codon. Sequences from the 3' end predict that at least three of the four clones do not have an intact, functional nef gene. All four cloned viruses yield infectious virus in HUT-78 and all four cloned viruses are cytopathic.
Animals↗
Comparison of simian immunodeficiency virus isolates.
Information on the extent of genetic variability among non-human primate lentiviruses related to human immunodeficiency virus (HIV) is sorely lacking. Here we describe the isolation of two molecular clones from the simian immunodeficiency virus (SIV) and their use to derive restriction endonuclease maps of five SIV isolates from rhesus macaques and one from a cynomolgus macaque. Although similar, all six viral isolates are readily distinguishable; the single isolate from a cynomolgus macaque is the most different. The restriction endonuclease map of one macaque isolate (SIVMAC-251) is identical to that published by others for STLV-IIIAGM of African green monkeys and for HTLV-IV of humans. Nucleotide sequences from the envelope region of cloned SIVMAC-251 have more than 99% identify to previously published sequences for STLV-IIIAGM (refs 2, 4) and HTLV-IV (ref. 4). These results and other observations provide strong evidence that isolates previously referred to as STLV-IIIAGM and HTLV-IV by others are not authentic, but were derived from cell cultures infected with SIVMAC-251.
Acquired Immunodeficiency Syndrome↗
Simian immunodeficiency virus from African green monkeys.
Simian immunodeficiency virus (SIV) was isolated from the total peripheral blood mononuclear cell population and the monocyte-macrophage adherent cell population of three seropositive green monkeys originating from Kenya. SIV from these African green monkeys (SIVagm) was isolated and continuously produced with the MOLT-4 clone 8 (M4C18) cell line but not with a variety of other cells including HUT-78, H9, CEM, MT-4, U937, and uncloned MOLT-4 cells. Once isolated, these SIVagm isolates were found to replicate efficiently in M4C18, SupT1, MT-4, U937, and Jurkat-T cells but much less efficiently if at all in HUT-78, H9, CEM, and MOLT-4 cells. The range of CD4+ cells fully permissive for replication of these SIVagm isolates thus differs markedly from that of previous SIV isolates from macaques (SIVmac). These SIVagm isolates had a morphogenesis and morphology like that of human immunodeficiency virus (HIV) and other SIV isolates. Antigens of SIVagm and SIVmac cross-reacted by comparative enzyme-linked immunosorbent assay only with reduced efficiency, and optimal results were obtained when homologous antibody and antigen were used. Western blotting (immunoblotting) of purified preparations of SIVagm isolate 385 (SIVagm385) revealed major viral proteins of 120, 27, and 16 kilodaltons (kDa). The presumed major core protein of 27 kDa cross-reacted antigenically with the corresponding proteins of SIVmac (28 kDa) and HIV-1 (24 kDa) by Western blotting. Hirt supernatant replicative-intermediate DNA prepared from cells freshly infected with SIVagm hybridized to SIVmac and HIV-2 DNA probes. Detection of cross-hybridizing DNA sequences, however, required very low stringency, and the restriction endonuclease fragmentation patterns of SIVagm were not similar to those of SIVmac and HIV-2. The nucleotide sequence of a portion of the pol gene of SIVagm385 revealed amino acid identities of 65% with SIVmac142, 64% with HIV-2ROD, and 56% with HIV-1BRU; SIVagm385 is thus related to but distinct from previously described primate lentiviruses SIVmac, HIV-1, and HIV-2. Precise information on the genetic makeup of these and other SIV isolates will possibly lead to better understanding of the history and evolution of these viruses and may provide insight into the origin of viruses that cause acquired immunodeficiency syndrome in humans.
Animals↗
Characterization of infectious molecular clones of simian immunodeficiency virus (SIVmac) and human immunodeficiency virus type 2: persistent infection of rhesus monkeys with molecularly cloned SIVmac.
Infection of macaque monkeys with simian immunodeficiency virus (SIV) is probably the best animal model currently available for studying acquired immunodeficiency syndrome. In this report, we describe three infectious molecular clones of SIVmac and one of human immunodeficiency virus type 2 (HIV-2) and their use in the study of cell and species specificity, animal infection, and the relationship of gene sequence to function. Replication of the cloned viruses in different cell lines varied dramatically. Some human CD4+ cell lines (HUT 78 and MT-4) supported the replication of SIVmac and HIV-2, while others (CEM and Jurkat-T) supported the replication of HIV-2 but not SIVmac. Growth of cloned virus in macaque lymphocytes in vitro was predictive of macaque infection in vivo. Macaque lymphocytes supported the replication of SIVmac239 and SIVmac251 but not SIVmac142 or HIV-2ROD. Using virus recovery and antibody response as criteria for infection, macaques that received cloned SIVmac251 and SIVmac239 became infected, while macaques receiving cloned SIVmac142 and HIV-2ROD did not become infected. Nucleotide sequences from the envelope region of all four cloned viruses demonstrated that there is considerable flexibility in the location of the translational termination (stop) signal. These infectious molecular clones will be very useful for future studies directed at the molecular basis for persistence, pathogenicity, tropism, and cell and species specificity.
Amino Acid Sequence↗
Mice transgenic for simian immunodeficiency virus nef are immunologically compromised.
An intact nef gene is essential for rapid development of immunodeficiency in human immunodeficiency virus and simian immunodeficiency virus infections. To assess the role of nef in the immune response, mice transgenic for SIV nef were constructed and the humoral and cellular immune response to herpes simplex virus type-1 (HSV-1), measured. Mice transgenic for SIVmac239 nef exhibited a significantly increased mortality rate when challenged with HSV-1 and also showed unusual antibody kinetics in response to viral challenge. During a 32-week period following exposure to HSV, it was noted that IgG subclass titers continued to rise in the nef+ animals, while titers of nef- animals decreased. Additionally, following secondary challenge with HSV, nef- mice had a significantly greater rise in HSV-neutralizing antibody titers than nef+ mice. A decreased proliferative response to the T cell mitogen, PHA, was noted in the nef+ animals. These results suggest that the presence of nef+ is sufficient to induce immune dysfunction.
3T3 Cells↗