CD8+ T cell-based strong selective pressure on multiple simian immunodeficiency virus targets in macaques possessing a protective MHC class I haplotype.

In human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections, host major histocompatibility complex class I (MHC-I) genotypes have a great impact on viral replication and MHC-I-associated viral genome mutations are selected under CD8+ T-cell pressure. Association of MHC-I genotypes with HIV/SIV control has been investigated at MHC-I allele levels but not fully at haplotype levels. We previously established groups of rhesus macaques sharing individual MHC-I haplotypes. In the present study, we compared viral genome diversification after SIV infection in macaques possessing a protective MHC-I haplotype, 90-010-Id, with those possessing a non-protective MHC-I haplotype, 90-010-Ie. These two MHC-I haplotypes are associated with immunodominant CD8+ T-cell responses targeting similar regions of viral Nef antigen. Analyses of viral genome sequences and antigen-specific T-cell responses showed four and two candidates of viral CD8+ T-cell targets associated with 90-010-Id and 90-010-Ie, respectively, in addition to the Nef targets. In these CD8+ T-cell target regions, higher numbers of mutations were detected at the setpoint after SIV infection in macaques possessing 90-010-Id than those possessing 90-010-Ie. These results indicate higher selective pressure on overall CD8+ T-cell targets associated with the protective MHC-I haplotype, suggesting a pattern of HIV/SIV control by multiple target-specific CD8+ T-cell responses.

Gene expression profiling reveals Nef induced deregulation of lipid metabolism in HIV-1 infected T cells.

Human Immunodeficiency Virus-1 (HIV-1) encodes a 27 kDa Negative Factor or Nef protein, which is increasingly proving to be a misnomer. Nef seems to be crucial for AIDS progression as individuals infected with nef-deleted strain of HIV were reported to become Long Term Non Progressors (LTNP). These findings necessitate tracing of Nef's footprint on landscape of cellular transcriptome favoring HIV-1 pathogenesis. We have tried to explore effect of Nef on cellular gene expression profile in conjunction with rest of HIV-1 proteins. Our results show that 237 genes are differentially regulated due to the presence of Nef during infection, which belong to several broad categories like "signaling", "apoptosis", "transcription" and "lipid metabolism" in gene ontology analysis. Furthermore, our results show that Nef causes disruption of lipid content in HIV-1 infected T cells. Molecular inhibitors of lipid metabolism like Atorvastatin and Ranolazine were found to have profound effect on wild type virus as compared to nef-deleted HIV-1. Thus our results suggest that interference in lipid metabolism is a potential mechanism through which Nef contributes in enhancing HIV-1 pathogenesis.

Escape from CD8(+) T cell responses in Mamu-B*00801(+) macaques differentiates progressors from elite controllers.

A small number of HIV-infected individuals known as elite controllers experience low levels of chronic phase viral replication and delayed progression to AIDS. Specific HLA class I alleles are associated with elite control, implicating CD8(+) T lymphocytes in the establishment of these low levels of viral replication. Most HIV-infected individuals that express protective HLA class I alleles, however, do not control viral replication. Approximately 50% of Mamu-B*00801(+) Indian rhesus macaques control SIVmac239 replication in the chronic phase in a manner that resembles elite control in humans. We followed both the immune response and viral evolution in SIV-infected Mamu-B*00801(+) animals to better understand the role of CD8(+) T lymphocytes during the acute phase of viral infection, when viral control status is determined. The virus escaped from immunodominant Vif and Nef Mamu-B*00801-restricted CD8(+) T lymphocyte responses during the critical early weeks of acute infection only in progressor animals that did not control viral replication. Thus, early CD8(+) T lymphocyte escape is a hallmark of Mamu-B*00801(+) macaques who do not control viral replication. By contrast, virus in elite controller macaques showed little evidence of variation in epitopes recognized by immunodominant CD8(+) T lymphocytes, implying that these cells play a role in viral control.

Nef decreases HIV-1 sensitivity to neutralizing antibodies that target the membrane-proximal external region of TMgp41.

Primate lentivirus nef is required for sustained virus replication in vivo and accelerated progression to AIDS. While exploring the mechanism by which Nef increases the infectivity of cell-free virions, we investigated a functional link between Nef and Env. Since we failed to detect an effect of Nef on the quantity of virion-associated Env, we searched for qualitative changes by examining whether Nef alters HIV-1 sensitivity to agents that target distinct features of Env. Nef conferred as much as 50-fold resistance to 2F5 and 4E10, two potent neutralizing monoclonal antibodies (nAbs) that target the membrane proximal external region (MPER) of TMgp41. In contrast, Nef had no effect on HIV-1 neutralization by MPER-specific nAb Z13e1, by the peptide inhibitor T20, nor by a panel of nAbs and other reagents targeting gp120. Resistance to neutralization by 2F5 and 4E10 was observed with Nef from a diverse range of HIV-1 and SIV isolates, as well as with HIV-1 virions bearing Env from CCR5- and CXCR4-tropic viruses, clade B and C viruses, or primary isolates. Functional analysis of a panel of Nef mutants revealed that this activity requires Nef myristoylation but that it is genetically separable from other Nef functions such as the ability to enhance virus infectivity and to downregulate CD4. Glycosylated-Gag from MoMLV substituted for Nef in conferring resistance to 2F5 and 4E10, indicating that this activity is conserved in a retrovirus that does not encode Nef. Given the reported membrane-dependence of MPER-recognition by 2F5 and 4E10, in contrast to the membrane-independence of Z13e1, the data here is consistent with a model in which Nef alters MPER recognition in the context of the virion membrane. Indeed, Nef and Glycosylated-Gag decreased the efficiency of virion capture by 2F5 and 4E10, but not by other nAbs. These studies demonstrate that Nef protects lentiviruses from one of the most broadly-acting classes of neutralizing antibodies. This newly discovered activity for Nef has important implications for anti-HIV-1 immunity and AIDS pathogenesis.

Inhibition of phagocytosis in HIV-1-infected macrophages relies on Nef-dependent alteration of focal delivery of recycling compartments.

Phagocytosis in macrophages is receptor mediated and relies on actin polymerization coordinated with the focal delivery of intracellular membranes that is necessary for optimal phagocytosis of large particles. Here we show that phagocytosis by various receptors was inhibited in primary human macrophages infected with wild-type HIV-1 but not with a nef-deleted virus. We observed no major perturbation of F-actin accumulation, but adaptor protein 1 (AP1)-positive endosome recruitment was inhibited in HIV-1-infected cells. Expression of negative factor (Nef) was sufficient to inhibit phagocytosis, and myristoylation as well as the LL and DD motifs involved in association of Nef with AP complexes were important for this inhibition. We observed that Nef interferes with AP1 in association with membranes and/or with a cleaved regulatory form of AP1. Finally, an alteration of the recruitment of vesicle-associated membrane protein (VAMP3)- and tumor necrosis factor-alpha (TNFalpha)-positive recycling endosomes regulated by AP1, but not of VAMP7-positive late endosomes, was observed in phagocytic cups of HIV-1-infected macrophages. We conclude that HIV-1 impairs optimal phagosome formation through Nef-dependent perturbation of the endosomal remodeling relying on AP1. We therefore identified a mechanism of macrophage function down-regulation in infected cells.

Host genetic determinants of T cell responses to the MRKAd5 HIV-1 gag/pol/nef vaccine in the step trial.

Understanding how human genetic variation impacts individual response to immunogens is fundamental for rational vaccine development. To explore host mechanisms involved in cellular immune responses to the MRKAd5 human immunodeficiency virus type 1 (HIV-1) gag/pol/nef vaccine tested in the Step trial, we performed a genome-wide association study of determinants of HIV-specific T cell responses, measured by interferon γ enzyme-linked immunospot assays. No human genetic variant reached genome-wide significance, but polymorphisms located in the major histocompatibility complex (MHC) region showed the strongest association with response to the HIV-1 Gag protein: HLA-B alleles known to be associated with differences in HIV-1 control were responsible for these associations. The implication of the same HLA alleles in vaccine-induced cellular immunity and in natural immune control is of relevance for vaccine design. Furthermore, our results demonstrate the importance of considering the host immunogenetic background in the analysis of immune responses to T cell vaccines.

Extralymphoid CD8+ T cells resident in tissue from simian immunodeficiency virus SIVmac239{Delta}nef-vaccinated macaques suppress SIVmac239 replication ex vivo.

Live-attenuated vaccination with simian immunodeficiency virus (SIV) SIVmac239Deltanef is the most successful vaccine product tested to date in macaques. However, the mechanisms that explain the efficacy of this vaccine remain largely unknown. We utilized an ex vivo viral suppression assay to assess the quality of the immune response in SIVmac239Deltanef-immunized animals. Using major histocompatibility complex-matched Mauritian cynomolgus macaques, we did not detect SIV-specific functional immune responses in the blood by gamma interferon (IFN-gamma) enzyme-linked immunospot assay at select time points; however, we found that lung CD8(+) T cells, unlike blood CD8(+) T cells, effectively suppress virus replication by up to 80%. These results suggest that SIVmac239Deltanef may be an effective vaccine because it elicits functional immunity at mucosal sites. Moreover, these results underscore the limitations of relying on immunological measurements from peripheral blood lymphocytes in studies of protective immunity to HIV/SIV.

Meta-analysis to test the association of HIV-1 nef amino acid differences and deletions with disease progression.

Previous relatively small studies have associated particular amino acid replacements and deletions in the HIV-1 nef gene with differences in the rate of HIV disease progression. We tested more rigorously whether particular nef amino acid differences and deletions are associated with HIV disease progression. Amino acid replacements and deletions in patients' consensus sequences were investigated for 153 progressor (P), 615 long-term nonprogressor (LTNP), and 2,311 unknown progressor sequences from 582 subtype B HIV-infected patients. LTNPs had more defective nefs (interrupted by frameshifts or stop codons), but on a per-patient basis there was no excess of LTNP patients with one or more defective nef sequences compared to the Ps (P = 0.47). The high frequency of amino acid replacement at residues S(8), V(10), I(11), A(15), V(85), V(133), N(157), S(163), V(168), D(174), R(178), E(182), and R(188) in LTNPs was also seen in permuted datasets, implying that these are simply rapidly evolving residues. Permutation testing revealed that residues showing the greatest excess over expectation (A(15), V(85), N(157), S(163), V(168), D(174), R(178), and R(188)) were not significant (P = 0.77). Exploratory analysis suggested a hypothetical excess of frameshifting in the regions (9)SVIG and (118)QGYF among LTNPs. The regions V(10) and (152)KVEEA of nef were commonly deleted in LTNPs. However, permutation testing indicated that none of the regions displayed significantly excessive deletion in LTNPs. In conclusion, meta-analysis of HIV-1 nef sequences provides no clear evidence of whether defective nef sequences or particular regions of the protein play a significant role in disease progression.

CD4 and MHC class 1 down-modulation activities of nef alleles from brain- and lymphoid tissue-derived primary HIV-1 isolates.

Human immunodeficiency virus type 1 (HIV-1) nef undergoes adaptive evolution in the central nervous system (CNS), reflecting altered requirements for HIV-1 replication in macrophages/microglia and brain-specific immune selection pressures. The role of Nef in HIV-1 neurotropism and pathogenesis of HIV-associated dementia (HAD) is unclear. In this study, we characterized 82 nef alleles cloned from brain, cerebral spinal fluid, spinal cord, and blood/lymphoid tissue-derived HIV-1 isolates from seven subjects with HAD. CNS isolate-derived nef alleles were genetically compartmentalized and had reduced sequence diversity compared to those from lymphoid tissue isolates. Defective nef alleles predominated in a brain-derived isolate from one of the seven subjects (MACS2-br). The ability of Nef to down-modulate CD4 and MHC class 1 (MHC-1) was generally conserved among nef alleles from both CNS and lymphoid tissues. However, the potency of CD4 and MHC-1 down-modulation was variable, which was associated with sequence alterations known to influence these Nef functions. These results suggest that CD4 and MHC-1 down-modulations are highly conserved functions among nef alleles from CNS- and lymphoid tissue-derived HIV-1 isolates that may contribute to viral replication and escape from immune surveillance in the CNS.

Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein.

Like other pathogenic viruses, HIV-1 down-modulates surface expression of major histocompatibility complex class I (MHC-I) molecules in infected cells, thus impairing lysis by cytotoxic T lymphocytes. We have observed that this phenomenon depends on the expression of Nef. nef is an early gene of primate lentiviruses, which is necessary for maintaining high virus loads and inducing AIDS. Nef is not necessary for viral replication in vitro and stimulates the endocytosis of CD4. We show that the expression of MHC-I at the surface of lymphoid, monocytic and epithelial cells was reduced in the presence of Nef protein from various HIV-1 strains. Whereas MHC-I protein synthesis and transport through the endoplasmic reticulum and cis Golgi apparatus occurred normally in Nef(+) cells, surface MHC-I molecules were rapidly internalized, accumulated in endosomal vesicles and were degraded. The stimulation of MHC-I endocytosis by Nef represents a previously undocumented viral mechanism for evading the immune response.

Association of simian immunodeficiency virus Nef with cellular serine/threonine kinases is dispensable for the development of AIDS in rhesus macaques.

The nef gene of simian immunodeficiency virus (SIV) is essential for high viral load and induction of AIDS in rhesus monkeys. A mutant form of the SIVmac239 Nef, which contains changes in a putative SH3-binding domain (amino acids 104 and 107 have been changed from PxxP to AxxA), does not associate with cellular serine/threonine kinases, but is fully active in CD4 downregulation and associates with the cellular tyrosine kinase Src. Infection of two rhesus macaques with SIVmac239 containing the mutant AxxA-Nef caused AIDS and rapid death in both animals. No reversions were observed in the majority of nef sequences analyzed from different time points during infection and from lymphatic tissues at the time of death. Our findings indicate that the putative SH3-ligand domain in SIVmac Nef and the association with cellular serine/threonine kinases are not important for efficient replication and pathogenicity of SIVmac in rhesus macaques.

HIV-1 Nef associated PAK and PI3-kinases stimulate Akt-independent Bad-phosphorylation to induce anti-apoptotic signals.

A highly conserved signaling property of Nef proteins encoded by human or simian immunodeficiency virus is the binding and activation of a PAK kinase whose function is unclear. Here we show that Nef-mediated p21-activated kinase (PAK) activation involves phosphatidylinositol 3-kinase, which acts upstream of PAK and is bound and activated by Nef similar to the manner of Polyoma virus middle T antigen. The Nef-associated phosphatidylinositol-3-PAK complex phosphorylated the pro-apoptotic Bad protein without involving the protein kinase B-Akt kinase, which is generally believed to inactivate Bad by serine phosphorylation. Consequently, Nef, but not a Nef mutant incapable of activating PAK, blocked apoptosis in T cells induced by serum starvation or HIV replication. Nef anti-apoptotic effects are likely a crucial mechanism for viral replication in the host and thus in AIDS pathogenesis.

Molecular and Genetic Characterization of Natural Variants of HIV-1 Nef Gene from North India and its Functional Implication in Down-Regulation of MHC-I and CD-4.

BACKGROUND: HIV-1 Nef is an important accessory protein with multiple effector functions. Genetic studies of the HIV-1 Nef gene show extensive genetic diversity and the functional studies have been carried out mostly with Nef derived from regions dominated by subtype B (North America & Europe). OBJECTIVE: This study was carried out to characterize genetic variations of the Nef gene from HIV-1 infected individuals from North India and to find out their functional implications. METHODS: The unique representative variants were sub-cloned in a eukaryotic expression vector and further characterized with respect to their ability to downregulate cell surface expression of CD4 and MHC-1 molecules. RESULTS: The phylogenetic analysis of Nef variants revealed sequence similarity with either consensus subtype B or B/C recombinants. Boot scan analysis of some of our variants showed homology to B/C recombinant and some to wild type Nef B. Extensive variations were observed in most of the variants. The dN/dS ratio revealed 80% purifying selection and 20% diversifying selection implying the importance of mutations in Nef variants. Intracellular stability of Nef variants differed greatly when compared with wild type Nef B and C. There were some variants that possessed mutations in the functional domains of Nef and responsible for its differential CD4 and MHC-1 downregulation activity. CONCLUSION: We observed enhanced biological activities in some of the variants, perhaps arising from amino acid substitutions in their functional domains. The CD4 and MHC-1 down-regulation activity of Nef is likely to confer immense survival advantage allowing the most rare genotype in a population to become the most abundant after a single selection event.

MS4--Multi-Scale Selector of Sequence Signatures: an alignment-free method for classification of biological sequences.

BACKGROUND: While multiple alignment is the first step of usual classification schemes for biological sequences, alignment-free methods are being increasingly used as alternatives when multiple alignments fail. Subword-based combinatorial methods are popular for their low algorithmic complexity (suffix trees ...) or exhaustivity (motif search), in general with fixed length word and/or number of mismatches. We developed previously a method to detect local similarities (the N-local decoding) based on the occurrences of repeated subwords of fixed length, which does not impose a fixed number of mismatches. The resulting similarities are, for some "good" values of N, sufficiently relevant to form the basis of a reliable alignment-free classification. The aim of this paper is to develop a method that uses the similarities detected by N-local decoding while not imposing a fixed value of N. We present a procedure that selects for every position in the sequences an adaptive value of N, and we implement it as the MS4 classification tool. RESULTS: Among the equivalence classes produced by the N-local decodings for all N, we select a (relatively) small number of "relevant" classes corresponding to variable length subwords that carry enough information to perform the classification. The parameter N, for which correct values are data-dependent and thus hard to guess, is here replaced by the average repetitivity kappa of the sequences. We show that our approach yields classifications of several sets of HIV/SIV sequences that agree with the accepted taxonomy, even on usually discarded repetitive regions (like the non-coding part of LTR). CONCLUSIONS: The method MS4 satisfactorily classifies a set of sequences that are notoriously hard to align. This suggests that our approach forms the basis of a reliable alignment-free classification tool. The only parameter kappa of MS4 seems to give reasonable results even for its default value, which can be a great advantage for sequence sets for which little information is available.

The importance of nef in the induction of human immunodeficiency virus type 1 replication from primary quiescent CD4 lymphocytes.

The viral regulatory gene, nef, is unique to the human immunodeficiency viruses (HIV) and their related primate lentiviruses. Expression of the nef gene has been shown to be essential to the maintenance of high levels of virus replication and the development of pathogenesis in the animal model of simian immunodeficiency virus (SIV) infection. In contrast to this in vivo model, the use of standard T cell culture systems to study nef function in vitro has produced a spectrum of contradictory results, and has failed to demonstrate a significant positive influence of nef on viral life cycle. We have developed a cell model to study regulation of HIV-1 replication that we believe reflects more accurately virus-cell interactions as they occur in vivo. Our experimental system used acute virus infection of purified, quiescent CD4 lymphocytes and subsequent induction of viral replication through T cell activation. With this cell model, NL4-3 virus clones with open and mutated nef reading frames were compared for replication competence. The clones with nef mutations showed reproducible and significant reductions in both rates of growth and maximal titers achieved. The degree of reduced replication was dependent on initial virus inoculum and the timing of T cell activation. The influence of nef was highly significant for induction of virus replication from a latent state within resting CD4 cells. Its effect was less apparent for virus infection of fully proliferating CD4 cells. This study demonstrates that nef confers a positive growth advantage to HIV-1 that becomes readily discernable in the primary cell setting of virus induction through T cell activation. The experimental cell model, which we describe here, provides not only a means to study nef function in vitro, but also provides important clues to the function of nef in HIV infection in vivo.

Cytotoxic T lymphocyte-based control of simian immunodeficiency virus replication in a preclinical AIDS vaccine trial.

Recently, encouraging AIDS vaccine trials in macaques have implicated cytotoxic T lymphocytes (CTLs) in the control of the simian human immunodeficiency virus SHIV89.6P that induces acute CD4(+) T cell depletion. However, none of these vaccine regimens have been successful in the containment of replication of the pathogenic simian immunodeficiency viruses (SIVs) that induce chronic disease progression. Indeed, it has remained unclear if vaccine-induced CTL can control SIV replication. Here, we show evidence suggesting that vaccine-induced CTLs control SIVmac239 replication in rhesus macaques. Eight macaques vaccinated with DNA-prime/Gag-expressing Sendai virus vector boost were challenged intravenously with SIVmac239. Five of the vaccinees controlled viral replication and had undetectable plasma viremia after 5 wk of infection. CTLs from all of these five macaques rapidly selected for escape mutations in Gag, indicating that vaccine-induced CTLs successfully contained replication of the challenge virus. Interestingly, analysis of the escape variant selected in three vaccinees that share a major histocompatibility complex class I haplotype revealed that the escape variant virus was at a replicative disadvantage compared with SIVmac239. These findings suggested that the vaccine-induced CTLs had "crippled" the challenge virus. Our results indicate that vaccine induction of highly effective CTLs can result in the containment of replication of a highly pathogenic immunodeficiency virus.

Downregulation of cell-surface CD4 expression by simian immunodeficiency virus Nef prevents viral super infection.

The nef gene product encoded by the mac239 proviral clone of simian immunodeficiency virus (SIV) markedly enhances viral replication and pathogenesis in vivo. We have used this biologically active nef isolate to examine the phenotype of Nef in retrovirally transduced human T cells in culture. SIV Nef is shown to dramatically inhibit cell-surface expression of the CD4 glycoprotein without significantly affecting the total steady-state level of cellular CD4. This downregulation of the cell-surface CD4 receptor for human immunodeficiency virus type 1 (HIV-1) infection correlated with the acquisition of resistance to superinfection by HIV-1. However, SIV Nef did not affect the level of gene expression directed by the HIV-1 long terminal repeat. It is hypothesized that downregulation of cell-surface CD4 by Nef facilitates the efficient release of infectious progeny virions and, hence, viral spread in vivo.

Severe immunodeficiency associated with a human immunodeficiency virus 1 NEF/3'-long terminal repeat transgene.

We have generated several transgenic mouse strains carrying a human immunodeficiency virus 1 (HIV-1) NEF/3' long terminal repeat (LTR) transgene under control of a T cell-specific promoter-enhancer element, showing a depletion of CD4+ T cells in the thymus and periphery. The immunological functions of the line with the most dramatic changes in lymphocyte populations, B6/338L, were analyzed in greater detail. The presence of the transgene in the heterozygous animal is associated with a dominant severe immunodeficiency. Older animals develop lymph-adenopathy and splenomegaly. CD4+CD8+ and CD4+CD8- single positive thymocytes already are depleted in these mice at the earliest stages in ontogeny, and peripheral T cells are reduced in frequency and present cell surface marker expression, which is characteristic for memory and activated T cells. The immunological response of B6/338L mice to several viral infections is also greatly impaired. Thus, the HIV-1 NEF/3' LTR as transgene in T cells can cause immunodeficiency and disease with striking similarities to a known retrovirus-induced immunodeficiency called murine AIDS (H. C. Morse III, S. K. Chattopadhyay, M. Makino, T. N. Frederickson, A. W. Hügin, and J. W. Hartley. 1992. AIDS. 6:607).

Inhibition of human immunodeficiency virus type 1 by triciribine involves the accessory protein nef.

Triciribine (TCN) is a tricyclic nucleoside that inhibits human immunodeficiency virus type 1 (HIV-1) replication by a unique mechanism not involving the inhibition of enzymes directly involved in viral replication. This activity requires the phosphorylation of TCN to its 5' monophosphate by intracellular adenosine kinase. New testing with a panel of HIV and simian immunodeficiency virus isolates, including low-passage-number clinical isolates and selected subgroups of HIV-1, multidrug resistant HIV-1, and HIV-2, has demonstrated that TCN has broad antiretroviral activity. It was active in cell lines chronically infected with HIV-1 in which the provirus was integrated into chromosomal DNA, thereby indicating that TCN inhibits a late process in virus replication. The selection of TCN-resistant HIV-1 isolates resulted in up to a 750-fold increase in the level of resistance to the drug. DNA sequence analysis of highly resistant isolate HIV-1(H10) found five point mutations in the HIV-1 gene nef, resulting in five different amino acid changes. DNA sequencing of the other TCN-resistant isolates identified at least one and up to three of the same mutations observed in isolate HIV-1(H10). Transfer of the mutations from TCN-resistant isolate HIV-1(H10) to wild-type virus and subsequent viral growth experiments with increasing concentrations of TCN demonstrated resistance to the drug. We conclude that TCN is a late-phase inhibitor of HIV-1 replication and that mutations in nef are necessary and sufficient for TCN resistance.

Evaluation of safety and efficacy of RNAi against HIV-1 in the human immune system (Rag-2(-/-)gammac(-/-)) mouse model.

RNA interference (RNAi) gene therapy against HIV-1 by stable expression of antiviral short hairpin RNAs (shRNAs) can potently inhibit viral replication in T cells. Recently, a mouse model with a human immune system (HIS) was developed that can be productively infected with HIV-1. In this in vivo model, in which Rag-2(-/-)gamma(c)(-/-) mice are engrafted with human CD34(+)CD38(-) hematopoietic precursor cells, we evaluated an anti-HIV RNAi gene therapy. Human hematopoietic stem cells were transduced with a lentiviral vector expressing an shRNA against the HIV-1 nef gene (shNef) or the control vector. We observed normal development of the different cell subsets of the immune system. However, although initial transduction efficiencies were similar for both vectors, a reduced percentage of transduced human immune cells was observed for the shNef vector after establishment of the HIS in vivo. Further studies are required to fully evaluate the safety implications. When we infected the mature human CD4(+) T cells from the HIS mouse ex vivo with HIV-1, potent inhibition of viral replication was scored in shNef-expressing cells, confirming efficacy. When challenged with an shNef-resistant HIV-1 variant, equal replication was scored in control and shNef-expressing cells, confirming sequence-specificity of the RNAi therapy. We thus demonstrated that an antiviral RNAi-based gene therapy on blood stem cells leads to HIV-1-resistant T cells in vivo, an important proof of concept in the clinical development of RNAi against HIV-1.