Vpr Mutants Research Articles

Dynamic disruptions in nuclear envelope architecture and integrity induced by HIV-1 Vpr.

Human immunodeficiency virus-1 (HIV-1) Vpr expression halts the proliferation of human cells at or near the G2 cell-cycle checkpoint. The transition from G2 to mitosis is normally controlled by changes in the state of phosphorylation and subcellular compartmentalization of key cell-cycle regulatory proteins. In studies of the intracellular trafficking of these regulators, we unexpectedly found that wild-type Vpr, but not Vpr mutants impaired for G2 arrest, induced transient, localized herniations in the nuclear envelope (NE). These herniations were associated with defects in the nuclear lamina. Intermittently, these herniations ruptured, resulting in the mixing of nuclear and cytoplasmic components. These Vpr-induced NE changes probably contribute to the observed cell-cycle arrest.

HIV-1 Vpr transactivates LTR-directed expression through sequences present within -278 to -176 and increases virus replication in vitro.

Human immunodeficiency virus type 1 (HIV-1) Vpr, a 14-kDa virion-associated protein, plays an important role in the viral life cycle. Using a panel of truncated HIV-1 LTR-CAT constructs and Vpr expression plasmid, we have identified sequences from nucleotide -278 to -176 in LTR as Vpr-mediated transactivation domain. This region includes the glucocorticoid response element (GRE) in HIV-1 LTR. Transactivation by Vpr was noted with the HIV-1 LTR reporter constructs containing CAT or luciferase. A similar effect was also observed with a construct in which the GRE motif was linked to CAT. Studies involving Vpr mutants identified that helical domains I and III, and amino acid residues at G75 and C76, are responsible for GRE-mediated LTR transactivation. The transactivation function of Vpr is independent of its cell cycle arrest activity. Further, viral replication studies indicated that Vpr-mediated increase in viral replication is directly correlated with the ability of Vpr to transactivate HIV-1 LTR. The results presented here demonstrate that Vpr activates HIV-1 LTR through the host GR pathway and suggest that an intact GRE in the LTR is critical for Vpr activity.

Nuclear export of human immunodeficiency virus type 1 Vpr is not required for virion packaging.

The human immunodeficiency virus type 1 Vpr protein is both packaged into virions and efficiently localized to the nucleus. In this report, we show that a significant fraction of Vpr also accumulates in the cytoplasm of virus-producing cells. Although Vpr shuttles between the nucleus and the cytoplasm, studies with an export-deficient Vpr mutant reveal that nuclear export is not required for virion incorporation.

A novel inducible expression system to study transdominant mutants of HIV-1 Vpr.

In this study, an episomal system for ecdysone-inducible gene expression was developed. Human embryonic kidney 293 cells (293VE) expressing a heterodimer of modified ecdysone and retinoid X receptors and the Epstein-Barr nuclear antigen-1 were screened. Plasmids containing the EBV replication origin, oriP, and the ecdysone-response element could replicate and persist in 293VE cells to inducibly express luciferase or Vpr. The induction level, tested with luciferase reporter plasmid, varied among cell lines from 254- to 2056-fold. In one highly inducible cell line, HIV-1 Vpr was expressed well and caused G2 cell cycle arrest in the presence of the inducer, while in the absence of the inducer, no Vpr protein or cell cycle arrest could be detected. Using different selection markers, HIV-1 Vpr was coexpressed with Vpr mutants defective in phosphorylation at Ser79 and G2 cell cycle arrest activity. These Vpr mutants were transdominant to wild-type Vpr for G2 cell cycle arrest activity, but did not alter wild-type Vpr phosphorylation. It is likely that the transdominant mutants and wild-type Vpr compete for a downstream target(s) of G2 cell cycle arrest.

Functional role of residues corresponding to helical domain II (amino acids 35 to 46) of human immunodeficiency virus type 1 Vpr.

Vpr, encoded by the human immunodeficiency virus type 1 genome, contains 96 amino acids and is a multifunctional protein with features which include cell cycle arrest at G(2), nuclear localization, participation in transport of the preintegration complex, cation channel activity, oligomerization, and interaction with cellular proteins, in addition to its incorporation into the virus particles. Recently, structural studies based on nuclear magnetic resonance and circular dichroism spectroscopy showed that Vpr contains a helix (HI)-turn-helix (HII) core at the amino terminus and an amphipathic helix (HIII) in the middle region. Though the importance of helical domains HI and HIII has been defined with respect to Vpr functions, the role of helical domain HII is not known. To address this issue, we constructed a series of mutants in which the HII domain was altered by deletion, insertion, and/or substitution mutagenesis. To enable the detection of Vpr, the sequence corresponding to the Flag epitope (DYKDDDDK) was added, in frame, to the Vpr coding sequences. Mutants, expressed through the in vitro transcription/translation system and in cells, showed an altered migration corresponding to deletions in Vpr. Substitution mutational analysis of residues in HII showed reduced stability for VprW38S-FL, VprL42G-FL, and VprH45W-FL. An assay involving cotransfection of NLDeltaVpr proviral DNA and a Vpr expression plasmid was employed to analyze the virion incorporation property of Vpr. Mutant Vpr containing deletions and specific substitutions (VprW38S-FL, VprL39G-FL, VprL42G-FL, VprG43P-FL, and VprI46G-FL) exhibited a negative virion incorporation phenotype. Further, mutant Vpr-FL containing deletions also failed to associate with wild-type Vpr, indicating a possible defect in the oligomerization feature of Vpr. Subcellular localization studies indicated that mutants VprDelta35-50-H-FL, VprR36W-FL, VprL39G-FL, and VprI46G-FL exhibited both cytoplasmic and nuclear localization, unlike other mutants and control Vpr-FL. While wild-type Vpr registered cell cycle arrest at G(2), mutant Vpr showed an intermediary effect with the exception of VprDelta35-50 and VprDelta35-50-H. These results suggest that residues in the HII domain are essential for Vpr functions.

Two putative alpha-helical domains of human immunodeficiency virus type 1 Vpr mediate nuclear localization by at least two mechanisms.

To identify the domains of Vpr that are involved nuclear localization, we transfected HeLa cells with a panel of expression vectors that encode mutant Vpr protein with deletions or substitutions within putative domains. Immunofluorescence staining of transfected cells revealed that wild-type Vpr was localized predominantly in the nucleus and the nuclear envelope and certainly in the cytoplasm. Introduction of substitutions or deletions within alphaH1 or alphaH2 resulted, by contrast, in diffuse expression over the entire cell. In addition, double mutations within both of these alpha-helical domains led to the complete absence of Vpr from nuclei. Next, we prepared HeLa cells that express chimeric proteins which consist of the alphaH1 and alphaH2 domains fused individually with green fluorescent protein (GFP) and a Flag tag and extracted them with digitonin and Triton X-100 prior to fixation. Flag-alphaH1-GFP was detected in the nucleus but not in the cytoplasm, while Flag-alphaH2-GFP was retained predominantly in the nucleus and in a small amount in the cytoplasm. The immunostaining patterns were almost eliminated by substitutions in each chimeric protein. Thus, it appeared that the two alpha-helical domains might be involved in nuclear import by binding to certain cellular factors. Taken together, our data suggest that the two putative alpha-helical domains mediate the nuclear localization of Vpr by at least two mechanisms.

Transdominant activity of human immunodeficiency virus type 1 Vpr with a mutation at residue R73.

The 96-amino-acid-long human immunodeficiency virus type 1 virion-encoded accessory protein Vpr is of particular interest, as this protein, which is found in association with viral particles, can exert a regulatory effect on both virus replication and host cell function. Evidently, Vpr, through interaction with several host regulatory proteins, can modulate transcription from the viral long terminal repeat promoter. Expression of Vpr in cells results in deregulation of cell proliferation during the cell cycle pathway at the G(2) stage. Vpr has unique structural features consisting of multiple functional domains. In this study, we have focused on the leucine/isoleucine-rich domain near the carboxyl terminus of Vpr at residue 73 (arginine) and have demonstrated that alterations at this residue result in ablation of transcriptional activity of Vpr and its ability to block cell cycle events at the G(2) stage. Interestingly, substitution mutations at R73 have resulted in a peptide with dominant negative activities on wild-type functions in transcription and host proliferation events. Results from in vitro and in vivo protein-protein interaction studies have revealed that functionally inactive mutant Vpr can be associated with wild-type protein, presumably through the N-terminal regions of the protein which have been shown to be important for Vpr oligomerization. Thus, it is likely that complexation of the mutant Vpr with wild-type protein functionally inactivates Vpr. The importance of these findings in light of the development of therapeutic strategies is discussed.

The HIV-1 virion-associated protein Vpr is a coactivator of the human glucocorticoid receptor: Clinical implications

AIDS 1s associated with immunosuppression. particularly of cellular or Thl-directed cellular Immunity and myopathy and muscular atrophy. These conditions are also seen as a result of exposure to elevated levels of glucocorticoids. The HIV-1 virion-associated accessory protein (Vpr) affects both viral replication and cellular transcription, proliferation and differentiation. We recently reported that Vpr enhances the activity of glucocotticoids in lymphoid and muscle-derived cell lines by interacting directly w!th the glucocorticnid receptor and general transcription factors, in particular TF-PB , acting as a coactivator. Vpr contains the signature motif w(LL also present in cellular nuclear receptor coactivators, such as SRC1 and p3OO/CBP, which mediates their interaction with the glucocorticoid and other nuclear hormone receptors. A mutant Vpr molecule wrth disruption of this coactivator signature motif lost its ability to influence transcription of glucocorticoid-responsrve genes and became a dominant negative inhibitor of Vpr. possibly by retaining its general transcnption factorbinding activities. The glucocorticord coactivator activity of Vpr may contribute to increased tissue glucocorticord sensitivity in the absence of hypercomsolism and to the pathogenesis of AIDS, specifically immunosupressron and myopathy and muscle atrophy. We speculate that Vpr might also participate rn the charactensttc lipodystrophy observed in HIV-infected patients receiving pmtease inhibitors (Kino T. et al 1999)

Cell cycle- and Vpr-mediated regulation of human immunodeficiency virus type 1 expression in primary and transformed T-cell lines.

Viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) transiently arrests cells in the G2 phase of the cell cycle and is a weak transcriptional transactivator. We found that Vpr increased HIV-1 long terminal repeat (LTR) activity in all cells examined but, when expressed at high levels, decreased HIV-1 LTR expression due to cytotoxic effects. Moreover, Vpr-mediated enhancement of HIV-1 LTR-driven transcription was observed in cycling primary human CD4(+) T cells but not in terminally differentiated, noncycling primary human macrophages. In single-round infection experiments using primary human CD4(+) T cells, proviral clones expressing either wild-type Vpr or Vpr mutants that retained the ability to cause a G2 arrest replicated to higher levels than proviruses lacking Vpr or expressing mutants of Vpr that did not cause an arrest. In support of the hypothesis that enhancement of HIV-1 LTR transcription by Vpr is an indirect effect of the ability of Vpr to delay cells in G2, counterflow centrifugal elutriation of cells into different phases of the cell cycle demonstrated that HIV-1 LTR expression was highest in G2. Finally, the ability of Vpr to upregulate viral transcription was dependent on a minimal promoter containing a functional TATA box and an enhancer.

Suppression of HIV-1 transcription and replication by a Vpr mutant.

Vpr, the 96 amino acid long protein represents one of the auxiliary proteins of human immunodeficiency virus type-1 (HIV-1), which exhibits the ability to increase the rate of replication of the virus in T cells. Structurally, this protein is composed of several regions such as the acidic domain with alpha helix at the amino terminus, leucine-isoleucine-rich domain (LR) near the carboxyl terminus and an arginine-rich domain at the C-terminus. Here, we evaluated the ability of wild-type and a spectrum of Vpr mutants with altered amino acid residues within the three major domains of Vpr to regulate of transcription of the HIV-1 LTR. Our results revealed that alterations of amino acids within the LR domain at position 73 from arginine to serine, renders Vpr defective in stimulating transcription of the viral promoter in human T-lymphocytic and astrocytic cells. Mutations within the N- and C-terminal domains had little or no effect on the transcriptional activity of Vpr. Of interest, ectopic expression of this mutant protein exerts a negative effect on the ability of wild-type Vpr, as well as the viral transactivator, Tat, in augmenting viral gene transcription. Production of the mutant Vpr interferes with the replication of the wild-type and delta Vpr virus in the cells. Accordingly, a Vpr mutant virus containing the transition of arginine to serine at position 73 exhibited an inhibitory effect on the replication of wild-type virus. Our results provide a new avenue for the utilization of the variant of the HIV-1 regulatory protein, Vpr, in suppressing replication of the viral genome in infected cells.

Mutational analysis of Vpr-induced G2 arrest, nuclear localization, and cell death in fission yeast.

Cell cycle G2 arrest, nuclear localization, and cell death induced by human immunodeficiency virus type 1 Vpr were examined in fission yeast by using a panel of Vpr mutations that have been studied previously in human cells. The effects of the mutations on Vpr functions were highly similar between fission yeast and human cells. Consistent with mammalian cell studies, induction of cell cycle G2 arrest by Vpr was found to be independent of nuclear localization. In addition, G2 arrest was also shown to be independent of cell killing, which only occurred when the mutant Vpr localized to the nucleus. The C-terminal end of Vpr is crucial for G2 arrest, the N-terminal alpha-helix is important for nuclear localization, and a large part of the Vpr protein is responsible for cell killing. It is evident that the overall structure of Vpr is essential for these cellular effects, as N- and C-terminal deletions affected all three cellular functions. Furthermore, two single point mutations (H33R and H71R), both of which reside at the end of each alpha-helix, disrupted all three Vpr functions, indicating that these two mutations may have strong effects on the overall Vpr structure. The similarity of the mutant effects on Vpr function in fission yeast and human cells suggests that fission yeast can be used as a model system to evaluate these Vpr functions in naturally occurring viral isolates.

The HIV-1 virion-associated protein vpr is a coactivator of the human glucocorticoid receptor.

The HIV-1 virion-associated accessory protein Vpr affects both viral replication and cellular transcription, proliferation, and differentiation. We report that Vpr enhances the activity of glucocorticoids in lymphoid and muscle-derived cell lines by interacting directly with the glucocorticoid receptor and general transcription factors, acting as a coactivator. Vpr contains the signature motif LXXLL also present in cellular nuclear receptor coactivators, such as steroid receptor coactivator 1 and p300/CREB-binding protein, which mediates their interaction with the glucocorticoid and other nuclear hormone receptors. A mutant Vpr molecule with disruption of this coactivator signature motif lost its ability to influence transcription of glucocorticoid-responsive genes and became a dominant-negative inhibitor of Vpr, possibly by retaining its general transcription factor-binding activities. The glucocorticoid coactivator activity of Vpr may contribute to increased tissue glucocorticoid sensitivity in the absence of hypercortisolism and to the pathogenesis of AIDS.

Interaction with the p6 domain of the gag precursor mediates incorporation into virions of Vpr and Vpx proteins from primate lentiviruses.

Vpr and Vpx proteins from human and simian immunodeficiency viruses (HIV and SIV) are incorporated into virions in quantities equivalent to those of the viral Gag proteins. We demonstrate here that Vpr and Vpx proteins from distinct lineages of primate lentiviruses were able to bind to their respective Gag precursors. The capacity of HIV type 1 (HIV-1) Vpr mutants to bind to Pr55(Gag) was correlated with their incorporation into virions. Molecular analysis of these interactions revealed that they required the C-terminal p6 domain of the Gag precursors. While the signal for HIV-1 Vpr binding lies in the leucine triplet repeat region of the p6 domain reported to be essential for incorporation, SIVsm Gag lacking the equivalent region still bound to SIVsm Vpr and Vpx, indicating that the determinants for Gag binding are located upstream of this region of the p6 domain. Binding to Gag cleavage products showed that HIV-1 Vpr interacted directly with the nucleocapsid protein (NC), whereas SIVsm Vpr and Vpx did not interact with NC but with the p6 protein. These results (i) reveal differences between HIV-1 and SIVsm for the p6 determinants required for Vpr and Vpx binding to Gag and (ii) suggest that HIV-1 Vpr and SIVsm Vpr and Vpx interact with distinct cleavage products of the precursor following proteolytic processing in the virions.

Human immunodeficiency virus type 1 vpr protein transactivation function: mechanism and identification of domains involved

The human immunodeficiency virus type 1 (HIV-1) Vpr protein is a virion-associated protein that localizes in the nucleus of infected cells. Vpr has been shown to facilitate HIV infection of non-dividing cells such as macrophages by contributing to the nuclear translocation of the pre-integration complex. More recently, Vpr expression has been shown to induce an accumulation of cells at the G2 phase of the cell-cycle. We have previously reported that Vpr stimulates reporter gene expression directed from the HIV-1 long terminal repeat (LTR) as well as from heterologous viral promoters. However, the mode of action of Vpr-mediated transactivation remains to be precisely defined. We report here that, for a constant amount of transfected DNA, the level of chloramphenicol acetyltransferase (CAT) mRNA is increased in Vpr-expressing cells using either HIV-1 or a murine leukemia virus (MLV) SL3-3 LTR-CAT reporter construct. Moreover, this Vpr-mediated transactivation requires that promoters direct a minimal level of basal expression. Our mutagenic analysis indicates that the transactivation mediated by Vpr is not dependent on the ability of the protein to localize in the nucleus or to be packaged in the virions. Interestingly, all transactivation-competent Vpr mutants were still able to induce a cell-cycle arrest. Conversely, transactivation-defective mutants lost the ability to mediate cell-cycle arrest, implying a functional relationship between these two functions. Overall, our results indicate that the G2 cell-cycle arrest mediated by Vpr creates a cellular environment where the HIV-1 LTR is transcriptionally more active.

Arginine Residues in the C-terminus of HIV-1 Vpr Are Important for Nuclear Localization and Cell Cycle Arrest

HIV-1 viral protein R (Vpr) is predominantly localized to the nucleus and plays an important role for viral preintegration complex import into the nucleus. In this study, we investigated the influence on subcellular localization of Arg residues in the C-terminus of Vpr. Consistent with previous studies, about 90% of the cells manifested diffuse nuclear staining in the Vpr-expressed cells. Besides diffuse nuclear staining, punctate perinuclear staining, and punctate cytoplasmic staining were also observed in the immunofluorescence studies. Deletion of the Ser-Arg-Ile-Gly residues (amino acids 79–82; SRIG) had no effect on the Vpr localization. However, deletion of the Arg-Gln-Arg-Arg residues (amino acids 85–88; RQRR) resulted in a smooth perinuclear staining pattern. Substitution of five Arg residues with Asn (amino acids 80, 85, 87, 88, and 90; R → N5) resulted in a diffuse cytoplasmic staining. Subcellular fractionation analyses support the immunofluorescence staining results. These findings indicate that the C-terminal Arg residues of HIV-1 Vpr play an important role for Vpr nuclear localization. All the Vpr mutants were appropriately expressed, exhibited no significant defect on the protein stability, and were incorporated efficiently into virus-like particles. Both SRIG and R → N5 mutants lost their cell cycle arrest activities and the RQRR deletion only exhibited a low level of cell arrest activity. Therefore, the Arg residues in the HIV-1 Vpr C-terminus are important for Vpr nuclear localization and cell cycle arrest , but had no effect on protein stability or Vpr incorporation into virus-like particles.

Nuclear import, virion incorporation, and cell cycle arrest/differentiation are mediated by distinct functional domains of human immunodeficiency virus type 1 Vpr.

The vpr gene product of human immunodeficiency virus type 1 (HIV-1) is a virion-associated protein that is essential for efficient viral replication in monocytes/macrophages. Vpr is primarily localized in the nucleus when expressed in the absence of other viral proteins. Vpr is packaged efficiently into viral particles through interactions with the p6 domain of the Gag precursor polyprotein p55gag. We developed a panel of expression vectors encoding Vpr molecules mutated in the amino-terminal helical domain, leucine-isoleucine (LR) domain, and carboxy-terminal domain to map the different functional domains and to define the interrelationships between virion incorporation, nuclear localization, cell cycle arrest, and differentiation functions of Vpr. We observed that substitution mutations in the N-terminal domain of Vpr impaired both nuclear localization and virion packaging, suggesting that the helical structure may play a vital role in modulating both of these biological properties. The LR domain was found to be involved in the nuclear localization of Vpr. In contrast, cell cycle arrest appears to be largely controlled by the C-terminal domain of Vpr. The LR and C-terminal domains do not appear to be essential for virion incorporation of Vpr. Interestingly, we found that two Vpr mutants harboring single amino acid substitutions (A30L and G75A) retained the ability to translocate to the nucleus but were impaired in the cell cycle arrest function. In contrast, mutation of Leu68 to Ser resulted in a protein that localizes in the cytoplasm while retaining the ability to arrest host cell proliferation. We speculate that the nuclear localization and cell cycle arrest functions of Vpr are not interrelated and that these functions are mediated by separable putative functional domains of Vpr.

Human immunodeficiency virus type 1 Vpr protein binds to the uracil DNA glycosylase DNA repair enzyme

The role of the accessory gene product Vpr during human immunodeficiency virus type 1 infection remains unclear. We have used the yeast two-hybrid system to identify cellular proteins that interact with Vpr and could be involved in its function. A cDNA clone which encodes the human uracil DNA glycosylase (UNG), a DNA repair enzyme involved in removal of uracil in DNA, has been isolated. Interaction between Vpr and UNG has been demonstrated by in vitro protein-protein binding assays using translated, radiolabeled Vpr and UNG recombinant proteins expressed as a glutathione S-transferase fusion protein. Conversely, purified UNG has been demonstrated to interact with Vpr recombinant protein expressed as a glutathione S-transferase fusion protein. Coimmunoprecipitation experiments confirmed that Vpr and UNG are associated within cells expressing Vpr. By using a panel of C- and N-terminally deleted Vpr mutants, we have determined that the core protein of Vpr, spanning amino acids 15 to 77, is involved in the interaction with UNG. We also demonstrate by in vitro experiments that the enzymatic activity of UNG is retained upon interaction with Vpr.

The glucocorticoid receptor type II complex is a target of the HIV-1 vpr gene product.

The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a 15-kDa virion-associated protein that functions as a regulator of cellular processes linked to the HIV life cycle. We report the interaction of a 41-kDa cytosolic viral protein R interacting protein 1 (Rip-1) with Vpr in vitro. Rip-1 displays a wide tissue distribution, including relevant targets of HIV infection. Vpr protein induced nuclear translocation of Rip-1, as did glucocorticoid receptor (GR)-II-stimulating steroids. Importantly, Vpr and Rip-1 coimmunoprecipitated with the human GR as part of an activated receptor complex. Vpr complementation of a vpr mutant virus was also mimicked by GR-II-stimulating steroids. Vpr and GR-II actions were inhibited by mifepristone, a GR-II pathway inhibitor. Together these data directly link the activity of the vpr gene product to the glucocorticoid steroid pathway and provide a biochemical mechanism for the cellular and viral activity of Vpr, as well as suggest that a unique class of antivirals, which includes mifepristone (RU486), may influence HIV-1 replication.

Functional Analysis of the vpx, vpr, and nef Genes of Simian Immunodeficiency Virus

The role of the vpx, vpr, and nef genes in the replication of simian immunodeficiency virus (SIV) was investigated using point and deletion mutations in these genes. The effects on replication kinetics of single or combined mutants--vpx, vpr, vpx-vpr, vpx-nef, vpr-nef, and vpx-vpr-nef--in established lymphoid CEMx174 and MT-4 cells were negligible, except that the postinfection appearance of vpx-nef, vpr-nef, and vpx-vpr-nef progeny virus was slightly delayed in MT-4 cells. The vpx, but not the vpr, point mutation reverted to wild-type sequences within 12 days after infection, suggesting that stronger selection pressure for Vpx than for Vpr expression might exist in these established cell lines. In contrast to growth in the lymphoid cell lines, replication of vpx-deleted viruses in macaque peripheral blood mononuclear cells (PBMC) was severely impaired, indicating that Vpx is necessary for efficient replication in PBMC. In contrast, the vpr mutant exhibited different degrees of impairment depending on the donor animal used as a source of PBMC. A virus encoding a Vpx-Vpr fusion protein replicated in PBMC comparably to a vpr deletion mutant virus, whereas a frameshift deletion at the vpx-vpr junction of this mutant eliminated virus replication, suggesting that deletion of the C-terminal half of Vpx was partially compensated by the presence of the large Vpr portion in the fusion protein. Deletion of the nef gene did not affect SIVmac replication in PBMC. The Vpx and Vpr proteins expressed in COS-1 cells were detected in the extracellular medium and did not crossreact with Vpr- and Vpx-specific antisera, in spite of extensive amino acid similarity between these proteins. These studies indicate the importance of Vpx and Vpr in SIVmac infection and suggest that these proteins are antigenically and functionally distinct.

Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques.

Adult macaques do not develop disease after infection with a nef deletion mutant of the simian immunodeficiency virus (SIV) and are protected against challenge with pathogenic virus. This finding led to the proposal to use nef-deleted viruses as live, attenuated vaccines to prevent human acquired immunodeficiency syndrome (AIDS). In contrast, neonatal macaques developed persistently high levels of viremia after oral exposure to and SIV nef, vpr, and negative regulatory element (NRE) deletion mutant. Severe hemolytic anemia, thrombocytopenia, and CD4+ T cell depletion were observed, indicating that neither nef nor vpr determine pathogenicity in neonates. Because such constructs have retained their pathogenic potential, they should not be used as candidate live, attenuated virus vaccines against human AIDS.