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Long interspersed element type 1 (LINE-1) is the only known type of retroelement that can replicate autonomously, and its retrotransposition activity can trigger interferon (IFN) production. IFN production suppresses the infectivity of exogenous viruses, such as human immunodeficiency virus (HIV). As a counteraction, HIV has been reported to use multiple proteins and mechanisms to suppress LINE-1 replication. However, the mechanisms of HIV-mediated LINE-1 regulation are not fully understood. In this study, we discovered that Nef protein, which is expressed by HIV and is important for HIV pathogenesis, inhibits LINE-1 retrotransposition. Two distinct mechanisms have been uncovered for Nef-induced LINE-1 suppression. Without direct interaction with LINE-1 DNA, Nef potently inhibits the promoter activity of the LINE-1 5'-untranslated region (5'-UTR) and reduces the expression levels of LINE-1 RNA and proteins. Alternatively, although Nef does not bind to the LINE-1 open reading frame 1 protein (ORF1p) or LINE-1 RNA, it significantly compromises the ORF1p-LINE-1 RNA interaction, which is essential for LINE-1 retrotransposition. Both mechanisms can be suppressed by the G2A mutation, which abolishes myristoylation of Nef, suggesting that membrane attachment is essential for Nef to suppress LINE-1. Consequently, through LINE-1 inhibition, Nef downregulates IFN production in host cells. Therefore, our data revealed that Nef is a potent LINE-1 suppressor and an effective innate immune regulator, which not only provides new information on the intricate interaction between HIV, LINE-1, and IFN signaling systems but also strengthens the importance of Nef in HIV infection and highlights the potential of designing novel Nef-targeting anti-HIV drugs. IMPORTANCE Human immunodeficiency viruses are pathogens of AIDS that were first discovered almost 40 years ago and continue to threaten human lives to date. While currently used anti-HIV drugs are sufficient to suppress viral loads in HIV-infected patients, both drug-resistant HIV strains and adverse side effects triggered by the long-term use of these drugs highlight the need to develop novel anti-HIV drugs targeting different viral proteins and/or different steps in viral replication. To achieve this, more information is required regarding HIV pathogenesis and especially its impact on cellular activities in host cells. In this study, we discovered that the Nef protein expressed by HIV potently inhibits LINE-1 retrotransposition. During our attempt to determine the mechanism of Nef-mediated LINE-1 suppression, two additional functions of Nef were uncovered. Nef effectively repressed the promoter activity of LINE-1 5'-UTR and destabilized the interaction between ORF1p and LINE-1 RNA. Consequently, Nef not only compromises LINE-1 replication but also reduces LINE-1-triggered IFN production. The reduction in IFN production, in theory, promotes HIV infectivity. Together with its previously known functions, these findings indicate that Nef is a potential target for the development of novel anti-HIV drugs. Notably, the G2 residue, which has been reported to be essential for most Nef functions, was found to be critical in the regulation of innate immune activation by Nef, suggesting that compromising myristoylation or membrane attachment of Nef may be a good strategy for the inhibition of HIV infection.

Most simian immunodeficiency viruses (SIVs) use Nef to counteract restriction by the tetherin proteins of their nonhuman primate hosts. In addition to counteracting tetherin, SIV Nef has a number of other functions, including the downmodulation of CD3, CD4, and major histocompatibility complex class I (MHC I) molecules from the surface of SIV-infected cells and the enhancement of viral infectivity by preventing the incorporation of SERINC5 into virions. Although these activities require different surfaces of Nef, they can be difficult to separate because of their dependence on similar interactions with AP-1 or AP-2 for clathrin-mediated endocytosis. We previously observed extensive overlap of the SIV Nef residues required for counteracting tetherin and SERINC5. Here, we define substitutions in Nef that separate anti-tetherin activity from SERINC5 antagonism and other activities of Nef. This information was used to engineer an infectious molecular clone of SIV (SIV mac 239 nef SA ) that is sensitive to tetherin but retains CD3, CD4, MHC I, and SERINC5 downmodulation. In primary rhesus macaque CD4 + T cells, SIV mac 239 nef SA exhibits impaired replication compared to wild-type SIV mac 239 under conditions of interferon-induced upregulation of tetherin. These results demonstrate that tetherin antagonism can be separated from other Nef functions and that resistance to tetherin is essential for optimal replication in primary CD4 + T cells. IMPORTANCE Tetherin is an interferon-inducible transmembrane protein that prevents the detachment of enveloped viruses from infected cells by physically tethering nascent virions to cellular membranes. SIV Nef downmodulates simian tetherin to overcome this restriction in nonhuman primate hosts. Nef also enhances virus infectivity by preventing the incorporation of SERINC5 into virions and contributes to immune evasion by downmodulating other proteins from the cell surface. To assess the contribution of tetherin antagonism to virus replication, we engineered an infectious molecular clone of SIV with substitutions in Nef that uncouple tetherin antagonism from other Nef functions. These substitutions impaired virus replication in interferon-treated macaque CD4 + T cells, revealing the impact of tetherin on SIV replication under physiological conditions in primary CD4 + lymphocytes.

Objective: HIV-associated CNS dysfunction is a significant problem among people with HIV (PWH), who now live longer due to viral suppression from combined anti-retroviral therapy (ART). Over the course of infection, HIV generates toxic viral proteins and induces inflammatory cytokines that have toxic effects on neurons in the CNS. Among these viral proteins, HIV Nef has been found in neurons of postmortem brain specimens from PWH. However, the source of Nef and its impact on neuronal cell homeostasis are still elusive., Methods and Results: Here, in using a simian immunodeficiency virus (SIV) infected rhesus macaque model of neuroHIV, we find SIV Nef reactivity in the frontal cortex, hippocampus and cerebellum of SIV-infected animals using immunohistochemistry (IHC). Interestingly, SIV-infected macaques treated with ART also showed frequent Nef positive cells in the cerebellum and hippocampus. Using dual quantitative RNAscope and IHC, we observed cells that were positive for Nef, but were not for SIV RNA, suggesting that Nef protein is present in cells that are not actively infected with SIV. Using cell specific markers, we observed Nef protein in microglia/macrophages and astrocytes. Importantly, we also identified a number of NeuN-positive neurons, which are not permissive to SIV infection, but contained Nef protein. Further characterization of Nef-positive neurons showed caspase 3 activation, indicating late stage apoptosis in the CNS neurons., Conclusions: Our results suggest that regardless of ART status, Nef is expressed in the brain of SIV infected macaques and may contribute to neurological complications seen in PWH., Competing Interests: The authors have declared that no competing interests exist.

Eukaryotic cells constitutively produce nanovesicles of 50-150 nm of diameter, referred to as exosomes, upon release of the contents of multivesicular bodies (MVBs). We recently characterized a novel, exosome-based way to induce cytotoxic T lymphocyte (CTL) immunization against full-length antigens. It is based on DNA vectors expressing products of fusion between the exosome-anchoring protein Nef mutant (Nef mut ) with the antigen of interest. The strong efficiency of Nef mut to accumulate in MVBs results in the production of exosomes incorporating huge amounts of the desired antigen. When translated in animals, the injection of Nef mut -based DNA vectors generates engineered exosomes whose internalization in antigen-presenting cells induces cross-priming and antigen-specific CTL immunity. Here, we describe the molecular strategies we followed to produce DNA vectors aimed at generating immunogenic exosomes potentially useful to elicit a CTL immune response against antigens expressed by the etiologic agents of major chronic viral infections, i.e., HIV-1, HBV, and the novel tumor-associated antigen HOXB7. Unique methods intended to counteract intrinsic RNA instability and nuclear localization of the antigens have been developed. The success we met with the production of these engineered exosomes opens the way towards pre-clinic experimentations devoted to the optimization of new vaccine candidates against major infectious and tumor pathologies.

The host restriction factor tetherin inhibits virion release from infected cells and poses a significant barrier to successful zoonotic transmission of primate lentiviruses to humans. While most simian immunodeficiency viruses (SIV), including the direct precursors of human immunodeficiency virus type 1 (HIV-1) and HIV-2, use their Nef protein to counteract tetherin in their natural hosts, they fail to antagonize the human tetherin ortholog. Pandemic HIV-1 group M and epidemic group O strains overcame this hurdle by adapting their Vpu and Nef proteins, respectively, whereas HIV-2 group A uses its envelope (Env) glycoprotein to counteract human tetherin. Whether or how the remaining eight groups of HIV-2 antagonize this antiviral factor has remained unclear. Here, we show that Nef proteins from diverse groups of HIV-2 do not or only modestly antagonize human tetherin, while their ability to downmodulate CD3 and CD4 is highly conserved. Experiments in transfected cell lines and infected primary cells revealed that not only Env proteins of epidemic HIV-2 group A but also those of a circulating recombinant form (CRF01_AB) and rare groups F and I decrease surface expression of human tetherin and significantly enhance progeny virus release. Intriguingly, we found that many SIVsmm Envs also counteract human as well as smm tetherin. Thus, Env-mediated tetherin antagonism in different groups of HIV-2 presumably stems from a preadaptation of their SIVsmm precursors to humans. In summary, we identified a phenotypic trait of SIVsmm that may have facilitated its successful zoonotic transmission to humans and the emergence of HIV-2. IMPORTANCE HIV-2 groups A to I resulted from nine independent cross-species transmission events of SIVsmm to humans and differ considerably in their prevalence and geographic spread. Thus, detailed characterization of these viruses offers a valuable means to elucidate immune evasion mechanisms and human-specific adaptations determining viral spread. In a systematic comparison of rare and epidemic HIV-2 groups and their simian SIVsmm counterparts, we found that the ability of Nef to downmodulate the primary viral entry receptor CD4 and the T cell receptor CD3 is conserved, while effects on CD28, CD74, and major histocompatibility complex class I surface expression vary considerably. Furthermore, we show that not only the Env proteins of HIV-2 groups A, AB, F, and I but also those of some SIVsmm isolates antagonize human tetherin. This finding helps to explain why SIVsmm has been able to cross the species barrier to humans on at least nine independent occasions., (Copyright © 2018 American Society for Microbiology.)

Several HIV-1 subtypes are co-circulating among various high-risk groups in China, and an increasing prevalence of CRF01_AE was observed among MSM (men who have sex with men) within recent years. Patients infected with CRF01_AE may experience a more rapid disease progression than patients infected with non-CRF01_AE; however, the underlying mechanisms remains elusive. HIV-1 Nef is a multifunctional protein and plays critical roles in viral pathogenesis. Nef downregulates CD4 and human leukocyte antigen (HLA) to promote viral transmission and escape from the host immune response. In this study, we investigated the CD4 downmodulation activity of Nef proteins isolated from HIV-1 CRF01_AE and analyzed a potential relationship of Nef's capacity to downregulate CD4 with disease progression. We found that the majority of these Nefs from HIV-1 CRF01_AE efficiently downregulated CD4; Nefs with weaker CD4 downmodulation activity tended to be associated with higher CD4 levels and lower viral loads. Further elucidation revealed that amino acid residues at positions 3, 168, and 169 of CRF01_AE Nefs were associated with the capacity to downregulate CD4. Our data suggest that the capacity of Nef-mediated CD4 downregulation is not the only determinant for controlling disease progression, and other host and viral factors should be considered to explain the rapid disease progression of patients infected with HIV-1 CRF01_AE., (© 2017 Wiley Periodicals, Inc.)

Eradication of the HIV-1 latent reservoir represents the current paradigm to developing a cure for AIDS. HIV-1 has evolved multiple mechanisms to evade CD8 T cell responses, including HIV-1 Nef-mediated downregulation of MHC-I from the surface of infected cells. Nef transcripts and protein are detectable in samples from aviremic donors, suggesting that Nef expression in latently HIV-1-infected CD4 T cells protects them from immune-mediated clearance. Here, we tested 4 small molecule inhibitors of HIV-1 Nef in an in vitro primary CD4 T cell latency model and measured the ability of autologous ex vivo or HIV-1 peptide-expanded CD8 T cells to recognize and kill latently infected cells as a function of inhibitor treatment. Nef inhibition enhanced cytokine secretion by autologous CD8 T cells against latently HIV-1-infected targets in an IFN-γ release assay. Additionally, CD8 T cell-mediated elimination of latently HIV-1-infected cells was significantly enhanced following Nef blockade, measured as a reduction in the frequency of infected cells and Gag protein in cultures following viral outgrowth assays. We demonstrate for the first time to our knowledge that Nef blockade, in combination with HIV-specific CD8 T cell expansion, might be a feasible strategy to target the HIV-1 latent reservoir that should be tested further in vivo.

Background: Vaccination with inactivated (killed) whole-virus particles has been used to prevent a wide range of viral diseases. However, for an HIV vaccine this approach has been largely negated due to inherent safety concerns, despite the ability of killed whole-virus vaccines to generate a strong, predominantly antibody-mediated immune response in vivo. HIV-1 Clade B NL4-3 was genetically modified by deleting the nef and vpu genes and substituting the coding sequence for the Env signal peptide with that of honeybee melittin signal peptide to produce a less virulent and more replication efficient virus. This genetically modified virus (gmHIV-1 NL4-3 ) was inactivated and formulated as a killed whole-HIV vaccine, and then used for a Phase I human clinical trial (Trial Registration: Clinical Trials NCT01546818). The gmHIV-1 NL4-3 was propagated in the A3.01 human T cell line followed by virus purification and inactivation with aldrithiol-2 and γ-irradiation. Thirty-three HIV-1 positive volunteers receiving cART were recruited for this observer-blinded, placebo-controlled Phase I human clinical trial to assess the safety and immunogenicity., Results: Genetically modified and killed whole-HIV-1 vaccine, SAV001, was well tolerated with no serious adverse events. HIV-1 NL4-3 -specific PCR showed neither evidence of vaccine virus replication in the vaccine virus-infected human T lymphocytes in vitro nor in the participating volunteers receiving SAV001 vaccine. Furthermore, SAV001 with adjuvant significantly increased the pre-existing antibody response to HIV-1 proteins. Antibodies in the plasma of vaccinees were also found to recognize HIV-1 envelope protein on the surface of infected cells as well as showing an enhancement of broadly neutralizing antibodies inhibiting tier I and II of HIV-1 B, D, and A subtypes., Conclusion: The killed whole-HIV vaccine, SAV001, is safe and triggers anti-HIV immune responses. It remains to be determined through an appropriate trial whether this immune response prevents HIV infection.

Invariant NKT (iNKT) cells are innate-like T cells that respond rapidly with a broad range of effector functions upon recognition of glycolipid Ags presented by CD1d. HIV-1 carries Nef- and Vpu-dependent mechanisms to interfere with CD1d surface expression, indirectly suggesting a role for iNKT cells in control of HIV-1 infection. In this study, we investigated whether iNKT cells can participate in the innate cell-mediated immune response to HIV-1. Infection of dendritic cells (DCs) with Nef- and Vpu-deficient HIV-1 induced upregulation of CD1d in a TLR7-dependent manner. Infection of DCs caused modulation of enzymes in the sphingolipid pathway and enhanced expression of the endogenous glucosylceramide Ag. Importantly, iNKT cells responded specifically to rare DCs productively infected with Nef- and Vpu-defective HIV-1. Transmitted founder viral isolates differed in their CD1d downregulation capacity, suggesting that diverse strains may be differentially successful in inhibiting this pathway. Furthermore, both iNKT cells and DCs expressing CD1d and HIV receptors resided in the female genital mucosa, a site where HIV-1 transmission occurs. Taken together, these findings suggest that innate iNKT cell sensing of HIV-1 infection in DCs is an early immune detection mechanism, which is independent of priming and adaptive recognition of viral Ag, and is actively targeted by Nef- and Vpu-dependent viral immune evasion mechanisms., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Nef proteins from all primate Lentiviruses, including the simian immunodeficiency virus of chimpanzees (SIVcpz), increase viral progeny infectivity. However, the function of Nef involved with the increase in viral infectivity is still not completely understood. Nonetheless, until now, studies investigating the functions of Nef from SIVcpz have been conducted in the context of the HIV-1 proviruses. In an attempt to investigate the role played by Nef during the replication cycle of an SIVcpz, a Nef-defective derivative was obtained from the SIVcpzWTGab2 clone by introducing a frame shift mutation at a unique restriction site within the nef sequence. This nef-deleted clone expresses an N-terminal 74-amino acid truncated peptide of Nef and was named SIVcpz-tNef. We found that the SIVcpz-tNef does not behave as a classic nef-deleted HIV-1 or simian immunodeficiency virus of macaques SIVmac. Markedly, SIVcpz-tNef progeny from both Hek-293T and Molt producer cells were completely non-infectious. Moreover, the loss in infectivity of SIVcpz-tNef correlated with the inhibition of Gag and GagPol processing. A marked accumulation of Gag and very low levels of reverse transcriptase were detected in viral lysates. Furthermore, these observations were reproduced once the tNef peptide was expressed in trans both in SIVcpzΔNef and HIV-1WT expressing cells, demonstrating that the truncated peptide is a dominant negative for viral processing and infectivity for both SIVcpz and HIV-1. We demonstrated that the truncated Nef peptide binds to GagPol outside the protease region and by doing so probably blocks processing of both GagPol and Gag precursors at a very early stage. This study demonstrates for the first time that naturally-occurring Nef peptides can potently block lentiviral processing and infectivity.

It has been estimated that human immunodeficiency virus type 1 originated from the zoonotic transmission of simian immunodeficiency virus (SIV) of chimpanzees, SIVcpz, and that SIVcpz emerged by the recombination of two lineages of SIVs in Old World monkeys (SIVgsn/mon/mus in guenons and SIVrcm in red-capped mangabeys) and SIVcpz Nef is most closely related to SIVrcm Nef. These observations suggest that SIVrcm Nef had an advantage over SIVgsn/mon/mus during the evolution of SIVcpz in chimpanzees, although this advantage remains uncertain. Nef is a multifunctional protein which downregulates CD4 and coreceptor proteins from the surface of infected cells, presumably to limit superinfection. To assess the possibility that SIVrcm Nef was selected by its superior ability to downregulate viral entry receptors in chimpanzees, we compared its ability to down-modulate viral receptor proteins from humans, chimpanzees and red-capped mangabeys with Nef proteins from eight other different strains of SIVs. Surprisingly, the ability of SIVrcm Nef to downregulate CCR5, CCR2B and CXCR6 was comparable to or lower than SIVgsn/mon/mus Nef, indicating that ability to down-modulate chemokine receptors was not the selective pressure. However, SIVrcm Nef significantly downregulates chimpanzee CD4 over SIVgsn/mon/mus Nefs. Our findings suggest the possibility that the selection of SIVrcm Nef by ancestral SIVcpz is due to its superior capacity to down-modulate chimpanzees CD4 rather than coreceptor proteins.

Virus-specific CD8(+) T-cell responses are crucial for the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. Multiple studies on HIV-infected individuals and SIV-infected macaques have indicated association of several major histocompatibility complex class I (MHC-I) genotypes with lower viral loads and delayed AIDS progression. Understanding of the viral control mechanism associated with these MHC-I genotypes would contribute to the development of intervention strategy for HIV control. We have previously reported a rhesus MHC-I haplotype, 90-120-Ia, associated with lower viral loads after SIVmac239 infection. Gag206-216 and Gag241-249 epitope-specific CD8(+) T-cell responses have been shown to play a central role in the reduction of viral loads, whereas the effect of Nef-specific CD8(+) T-cell responses induced in all the 90-120-Ia(+) macaques on SIV replication remains unknown. Here, we identified three CD8(+) T-cell epitopes, Nef9-19, Nef89-97, and Nef193-203, associated with 90-120-Ia. Nef9-19 and Nef193-203 epitope-specific CD8(+) T-cell responses frequently selected for mutations resulting in viral escape from recognition by these CD8(+) T cells, indicating that these CD8(+) T cells exert strong suppressive pressure on SIV replication. Results would be useful for elucidation of the viral control mechanism associated with 90-120-Ia., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Local synthesis of proteins in the axons participates in axonogenesis and axon guidance to establish appropriate synaptic connections and confer plasticity. To study the transcripts present in the growth cones and axonal shafts of cultured rat hippocampal neurons, two chip devices, differing in their abilities to support axonal growth and branching, are designed and employed here to isolate large quantities of axonal materials. Cone-, shaft- and axon-residing transcripts with amounts higher than that of a somatodendritic transcript, Actg1 (γ-actin), are selected and classified. Since the chips are optically transparent, distribution of transcripts over axons can be studied by fluorescence in situ hybridization. Three transcripts, Cadm1 (cell adhesion molecule 1), Nefl (neurofilament light polypeptide), and Cfl1 (non-muscle cofilin) are confirmed to be preferentially localized to the growth cones, while Pfn2 (profilin2) is preferentially localized to the shafts of those axons growing on the chip that restricts axonal growth. The different growing conditions of axons on chips and on conventional coverslips do not affect the cone-preferred localization of Cadm1 and shaft-preferred localization of Pfn2, but affect the distributions of Nefl and Cfl1 over the axons at 14th day in vitro. Furthermore, the distributions of Cadm1 and Nefl over the axons growing on conventional coverslips undergo changes during in vitro development. Our results suggest a dynamic nature of the mechanisms regulating the distributions of transcripts in axonal substructures in a manner dependent upon both growth conditions and neuronal maturation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Unlabelled: Broadly targeted cellular immune responses are thought to be important for controlling replication of human and simian immunodeficiency viruses (HIV and SIV). However, eliciting such responses by vaccination is complicated by immunodominance, the preferential targeting of only a few of the many possible epitopes of a given antigen. This phenomenon may be due to the coexpression of dominant and subdominant epitopes by the same antigen-presenting cell and may be overcome by distributing these sequences among several different vaccine constructs. Accordingly, we tested whether vaccinating rhesus macaques with "minigenes" encoding fragments of Gag, Vif, and Nef resulted in broadened cellular responses capable of controlling SIV replication. We delivered these minigenes through combinations of recombinant Mycobacterium bovis BCG (rBCG), electroporated recombinant DNA (rDNA) along with an interleukin-12 (IL-12)-expressing plasmid (EP rDNA plus pIL-12), yellow fever vaccine virus 17D (rYF17D), and recombinant adenovirus serotype 5 (rAd5). Although priming with EP rDNA plus pIL-12 increased the breadth of vaccine-induced T-cell responses, this effect was likely due to the improved antigen delivery afforded by electroporation rather than modulation of immunodominance. Indeed, Mamu-A*01(+) vaccinees mounted CD8(+) T cells directed against only one subdominant epitope, regardless of the vaccination regimen. After challenge with SIVmac239, vaccine efficacy was limited to a modest reduction in set point in some of the groups and did not correlate with standard T-cell measurements. These findings suggest that broad T-cell responses elicited by conventional vectors may not be sufficient to substantially contain AIDS virus replication., Importance: Immunodominance poses a major obstacle to the generation of broadly targeted, HIV-specific cellular responses by vaccination. Here we attempted to circumvent this phenomenon and thereby broaden the repertoire of SIV-specific cellular responses by vaccinating rhesus macaques with minigenes encoding fragments of Gag, Vif, and Nef. In contrast to previous mouse studies, this strategy appeared to minimally affect monkey CD8(+) T-cell immundominance hierarchies, as seen by the detection of only one subdominant epitope in Mamu-A*01(+) vaccinees. This finding underscores the difficulty of inducing subdominant CD8(+) T cells by vaccination and demonstrates that strategies other than gene fragmentation may be required to significantly alter immunodominance in primates. Although some of the regimens tested here were extremely immunogenic, vaccine efficacy was limited to a modest reduction in set point viremia after challenge with SIVmac239. No correlates of protection were identified. These results reinforce the notion that vaccine immunogenicity does not predict control of AIDS virus replication., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Despite the use of siRNA in the downregulation of HIV-1 replication which has been reported, CD4 T lymphocytes are difficult to transfect with non-viral vectors. We determined whether second generation carbosilane dendrimers (2G-NN16 and 2G-03NN24) may be efficient transfectants in CD4 T lymphocytes. Dendrimers were also tested on macrophages to determine whether they can modify macrophage phenotype and induce an inflammatory response. The nanoconjugate formed by 2G-03NN24/siRNA-Nef presents the highest inhibition of HIV-1 replication. Dendrimers presented safety properties because they did not induce proliferation on CD4 T lymphocytes and decrease the release of TNFα and IL-12p40 by macrophages. Both dendrimers also decrease the phagocytosis activity. Additionally, 2G-03NN24 dendrimer decreases the CCL2 and CCR2 expression in macrophages. Carbosilane dendrimers 2G-NN16 and 2G-03NN24 can be used as efficient non-viral vectors for gene therapy applications, mainly in the treatment of HIV infection., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Unlabelled: Human immunodeficiency virus type 1 (HIV-1) Nef enhances the infectivity of progeny virions. However, Nef is dispensable for the production of HIV-1 virions of optimal infectivity if the producer cells are superinfected with certain gammaretroviruses. In the case of the ecotropic Moloney murine leukemia virus (M-MLV), the Nef-like effect is mediated by the glycosylated Gag (glycoGag) protein. We now show that the N-terminal intracellular domain of the type II transmembrane protein glycoGag is responsible for its effect on HIV-1 infectivity. In the context of a fully active minimal M-MLV glycoGag construct, truncations of the cytoplasmic domain led to a near total loss of activity. Furthermore, the cytoplasmic domain of M-MLV glycoGag was fully sufficient to transfer the activity to an unrelated type II transmembrane protein. Although the intracellular region of glycoGag is relatively poorly conserved even among ecotropic and xenotropic MLVs, it was also fully sufficient for the rescue of nef-deficient HIV-1 when derived from a xenotropic virus. A mutagenic analysis showed that only a core region of the intracellular domain that exhibits at least some conservation between murine and feline leukemia viruses is crucial for activity. In particular, a conserved YXXL motif in the center of this core region was critical. In addition, expression of the μ2 subunit of the AP-2 adaptor complex in virus producer cells was essential for activity. We conclude that the ability to enhance HIV-1 infectivity is a conserved property of the MLV glycoGag cytoplasmic domain and involves AP-2-mediated endocytosis., Importance: The Nef protein of HIV-1 and the entirely unrelated glycosylated Gag (glycoGag) protein of a murine leukemia virus (MLV) similarly enhance the infectiousness of HIV-1 particles by an unknown mechanism. MLV glycoGag is an alternative version of the structural viral Gag protein with an extra upstream region that provides a cytosolic domain and a plasma membrane anchor. We now show for the first time that the cytosolic domain of MLV glycoGag contains all the information needed to enhance HIV-1 infectivity and that this function of the cytosolic domain is conserved despite limited sequence conservation. Within the cytosolic domain, a motif that resembles a cellular sorting signal is critical for activity. Furthermore, the enhancement of HIV-1 infectivity depends on an endocytic cellular protein that is known to interact with such sorting signals. Together, our findings implicate the endocytic machinery in the enhancement of HIV-1 infectivity by MLV glycoGag.

Simian immunodeficiency virus (SIVsmm) infection of sooty mangabeys (Cercocebus atys) is characterized by stable CD4(+) T cell counts despite high plasma levels of CCR5-tropic viruses. However, in rare instances, SIVsmm acquires CXCR4 coreceptor tropism and causes severe CD4(+) T cell depletion, albeit without clinical signs of immunodeficiency. Here, we show that CXCR4-tropic SIVsmm strains lost their ability to downmodulate TCR-CD3 by evolving unusual Nef mutations that initially reduced (I132V) and subsequently disrupted (I123L and L146F) interaction with the CD3 ζ chain. This coevolution of Env and Nef function suggests that CD3 downmodulation is advantageous for viral replication in activated CCR5(+) memory T cells, but not in resting naive CXCR4(+) T cells that have not yet undergone TCR-CD3-mediated stimulation. This may explain why HIV-1, which generally lacks the CD3 downmodulation function, commonly switches to CXCR4 usage, whereas this is extremely rare for SIV strains that have retained this Nef activity., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The HIV-2 env's 3' end encodes the cytoplasmic tail (CT) of the Env protein. This genomic region also encodes the rev, Tat and Nef protein in overlapping reading frames. We studied the variability in the CT coding region in 46 clinical specimens and in 2 reference strains by sequencing and by culturing. The aims were to analyse the variability of Env CT and the evolution of proteins expressed from overlapping coding sequences., Results: A 70% reduction of the length of the CT region affected the HIV-2 ROD and EHO strains in vitro due to a premature stop codon in the env gene. In clinical samples this wasn't observed, but the CT length varied due to insertions and deletions. We noted 3 conserved and 3 variable regions in the CT. The conserved regions were those containing residues involved in Env endocytosis, the potential HIV-2 CT region implicated in the NF-kB activation and the potential end of the lentiviral lytic peptide one. The variable regions were the potential HIV-2 Kennedy region, the potential lentiviral lytic peptide two and the beginning of the potential lentiviral lytic peptide one. A very hydrophobic region was coded downstream of the premature stop codon observed in vitro, suggesting a membrane spanning region. Interestingly, the nucleotides that are responsible for the variability of the CT don't impact rev and Nef. However, in the Kennedy-like coding region variability resulted only from nucleotide changes that impacted Env and Tat together., Conclusion: The HIV-2 Env, Tat and Rev C-terminal part are subject to major length variations in both clinical samples and cultured strains. The HIV-2 Env CT contains variable and conserved regions. These regions don't affect the rev and Nef amino acids composition which evolves independently. In contrast, Tat co-evolves with the Env CT.

An effective vaccine remains the best solution to stop the spread of human immunodeficiency virus (HIV). Cellular immune responses have been repeatedly associated with control of viral replication and thus may be an important element of the immune response that must be evoked by an efficacious vaccine. Recombinant viral vectors can induce potent T-cell responses. Although several viral vectors have been developed to deliver HIV genes, only a few have been advanced for clinical trials. The live-attenuated yellow fever vaccine virus 17D (YF17D) has many properties that make it an attractive vector for AIDS vaccine regimens. YF17D is well tolerated in humans and vaccination induces robust T-cell responses that persist for years. Additionally, methods to manipulate the YF17D genome have been established, enabling the generation of recombinant (r)YF17D vectors carrying genes from unrelated pathogens. Here, we report the generation of seven new rYF17D viruses expressing fragments of simian immunodeficiency virus (SIV)mac239 Gag, Nef, and Vif. Studies in Indian rhesus macaques demonstrated that these live-attenuated vectors replicated in vivo, but only elicited low levels of SIV-specific cellular responses. Boosting with recombinant Adenovirus type-5 (rAd5) vectors resulted in robust expansion of SIV-specific CD8(+) T-cell responses, particularly those targeting Vif. Priming with rYF17D also increased the frequency of CD4(+) cellular responses in rYF17D/rAd5-immunized macaques compared to animals that received rAd5 only. The effect of the rYF17D prime on the breadth of SIV-specific T-cell responses was limited and we also found evidence that some rYF17D vectors were more effective than others at priming SIV-specific T-cell responses. Together, our data suggest that YF17D - a clinically relevant vaccine vector - can be used to prime AIDS virus-specific T-cell responses in heterologous prime boost regimens. However, it will be important to optimize rYF17D-based vaccine regimens to ensure maximum delivery of all immunogens in a multivalent vaccine.

Aim: To evaluate if the lentiviral accessory protein Nef can down-regulate the C-X-C chemokine receptor type 4 (CXCR4) in tumor cells and affect tumor cell proliferation, migration and angiogenesis., Materials and Methods: HeLa-(ACC) cells, which according to genotype analysis are virtually identical to the cervical cancer-derived HeLa cell line, were transfected with Nef from SIV(mac239) and expression levels of cell surface CXCR4 were monitored by flow cytometry. Real-time proliferation and migration of cells was measured with the xCELLigence system or with the in vitro scratch assay. In vitro tube formation was deployed to assess the effect of Nef on angiogenesis., Results: Cell surface down-regulation of CXCR4 was observed in HeLa-(ACC) cells after Nef transfection, as well as in the monkey kidney-derived COS-7 cell line after co-transfection of CXCR4 and Nef. Proliferation, as well as migration, of Nef-transfected HeLa-(ACC) cells appeared to be significantly reduced. In vitro tube formation was markedly lowered after Nef transfection, and CXCR4 knockdown with siRNA., Conclusion: SIV-Nef could serve as an interesting tool to study the biological behavior of CXCR4-expressing tumor cells and could be helpful in the discovery of new therapeutic approaches for the treatment of CXCR4-positive tumors.

Nef is one of the accessory proteins of human immunodeficiency viruses. Here, we noted that the relative expression level of Nef(NL4-3) is much lower than that of NefJR-CSF in HEK293 cells. By evaluating the expression level using a Nef mutant, it was indicated that amino acids 129-206 of Nef(NL4-3), that is, the C-terminal region named NLAA129-206, could contain the region responsible for the induction of the low protein expression level. In addition, the expression levels of the enhanced green fluorescent protein and Renilla luciferase became extremely low with the fusion of NLAA129-206. Interestingly, the NLAA129-206-corresponding sequences of other Nef variants with relatively high expression levels also induced the extremely low protein expression level by fusion. These results suggest that the C-terminal region of Nef can generally induce an extremely low protein expression level. Here, we propose that the C-terminal region of Nef could become an excellent tool for the induction of an extremely low expression level of arbitrary proteins by attachment as fusion proteins., (Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.)

The Step Trial showed that the MRKAd5 HIV-1 subtype B Gag/Pol/Nef vaccine did not protect men from HIV infection or reduce setpoint plasma viral RNA (vRNA) levels but, unexpectedly, it did modestly enhance susceptibility to HIV infection in adenovirus type 5 (Ad5)-seropositive, uncircumcised men. As part of the process to understand the results of the Step Trial, we designed a study to determine whether rhesus macaques chronically infected with a host-range mutant Ad5 (Ad5hr) and then immunized with a replication defective Ad5 SIVmac239 Gag/Pol/Nef vaccine were more resistant or susceptible to SIV infection than unimmunized rhesus macaques challenged with a series of escalating dose penile exposures to SIVmac 251. The Ad5 SIV vaccine induced CD8(+) T cell responses in 70% of the monkeys, which is similar to the proportion of humans that responded to the vaccine in the Step Trial. However, the vaccine did not protect vaccinated animals from penile SIV challenge. At the lowest SIV exposure dose (10(3) 50% tissue culture infective doses), 2 of 9 Ad5-seropositive animals immunized with the Ad5 SIV vaccine became infected compared to 0 of 34 animals infected in the other animal groups (naive animals, Ad5-seropositive animals immunized with the empty Ad5 vector, Ad5-seronegative animals immunized with the Ad5 SIV vaccine, and Ad5-seronegative animals immunized with the empty Ad5 vector). Penile exposure to more concentrated virus inocula produced similar rates of infection in all animal groups. Although setpoint viral loads were unaffected in Step vaccinees, the Ad5 SIV-immunized animals had significantly lower acute-phase plasma vRNA levels compared to unimmunized animals. Thus, the results of the nonhuman primate (NHP) study described here recapitulate the lack of protection against HIV acquisition seen in the Step Trial and suggest a greater risk of infection in the Ad5-seropositive animals immunized with the Ad5 SIV vaccine. Further studies are necessary to confirm the enhancement of virus acquisition and to discern associated mechanisms.

It is well established that the Nef proteins of human and simian immunodeficiency viruses (HIV and SIV) modulate major histocompatibility complex class I (MHC-I) cell surface expression to protect infected cells against lysis by cytotoxic T lymphocytes (CTLs). Recent data supported the observation that Nef also manipulates CTLs directly by down-modulating CD8αβ (J. A. Leonard, T. Filzen, C. C. Carter, M. Schaefer, and K. L. Collins, J. Virol. 85:6867-6881, 2011), but it remained unknown whether this Nef activity is conserved between different lineages of HIV and SIV. In this study, we examined a total of 42 nef alleles from 16 different primate lentiviruses representing most major lineages of primate lentiviruses, as well as nonpandemic HIV-1 strains and the direct precursors of HIV-1 (SIVcpz and SIVgor). We found that the vast majority of these nef alleles strongly down-modulate CD8β in human T cells. Primate lentiviral Nefs generally interacted specifically with the cytoplasmic tail of CD8β, and down-modulation of this receptor was dependent on the conserved dileucine-based motif and two adjacent acidic residues (DD/E) in the C-terminal flexible loop of SIV Nef proteins. Both of these motifs are known to be important for the interaction of HIV-1 Nef with AP-2, and they were also shown to be critical for down-modulation of CD4 and CD28, but not MHC-I, by SIV Nefs. Our results show that down-modulation of CD4, CD8β, and CD28 involves largely overlapping (but not identical) domains and is most likely dependent on conserved interactions of primate lentiviral Nefs with cellular adaptor proteins. Furthermore, our data demonstrate that Nef-mediated down-modulation of CD8αβ is a fundamental property of primate lentiviruses and suggest that direct manipulation of CD8+ T cells plays a relevant role in viral immune evasion.

Recombinant strains of replication-competent rhesus monkey rhadinovirus (RRV) were constructed in which strong promoter/enhancer elements were used to drive expression of simian immunodeficiency virus (SIV) Env or Gag or a Rev-Tat-Nef fusion protein. Cultured rhesus monkey fibroblasts infected with each recombinant strain were shown to express the expected protein. Three RRV-negative and two RRV-positive rhesus monkeys were inoculated intravenously with a mixture of these three recombinant RRVs. Expression of SIV Gag was readily detected in lymph node biopsy specimens taken at 3 weeks postimmunization. Impressive anti-SIV cellular immune responses were elicited on the basis of major histocompatibility complex (MHC) tetramer staining and gamma interferon enzyme-linked immunospot (ELISPOT) assays. Responses were much greater in magnitude in the monkeys that were initially RRV negative but were still readily detected in the two monkeys that were naturally infected with RRV at the time of immunization. By 3 weeks postimmunization, responses measured by MHC tetramer staining in the two Mamu-A*01(+) RRV-negative monkeys reached 9.3% and 13.1% of all CD8(+) T cells in peripheral blood to the Gag CM9 epitope and 2.3% and 7.3% of all CD8(+) T cells in peripheral blood to the Tat SL8 epitope. Virus-specific CD8(+) T cell responses persisted at high levels up to the time of challenge at 18 weeks postimmunization, and responding cells maintained an effector memory phenotype. Despite the ability of the RRVenv recombinant to express high levels of Env in cultured cells, and despite the appearance of strong anti-RRV antibody responses in immunized monkeys, anti-Env antibody responses were below our ability to detect them. Immunized monkeys, together with three unimmunized controls, were challenged intravenously with 10 monkey infectious doses of SIVmac239. All five immunized monkeys and all three controls became infected with SIV, but peak viral loads were 1.2 to 3.0 log(10) units lower and chronic-phase viral loads were 1.0 to 3.0 log(10) units lower in immunized animals than the geometric mean of unimmunized controls. These differences were statistically significant. Anti-Env antibody responses following challenge indicated an anamnestic response in the vaccinated monkeys. These findings further demonstrate the potential of recombinant herpesviruses as preventive vaccines for AIDS. We hypothesize that this live, replication-competent, persistent herpesvirus vector could match, or come close to matching, live attenuated strains of SIV in the degree of protection if the difficulty with elicitation of anti-Env antibody responses can be overcome.

The effectiveness of recombinant Adenovirus serotype 5 (Ad5) vectors to induce immune responses against targeted antigens has been limited by the presence of pre-existing or Ad5 vaccine induced anti-vector immunity. The Ad5 [E1-, E2b-] platform, a recombinant Ad5 with additional deletions, has been previously reported by us to induce immune responses in the presence of Ad5 immunity. In an Ad5 immune non-human primate (NHP) model, an Ad5 [E1-, E2b-] construct expressing HIV-1 Gag induced immune responses in the presence of pre-existing Ad5 immunity. In the present study we expand on these prior observations by comparing the cell mediated immune (CMI) responses induced by Ad5 [E1-, E2b-]-SIV-gag/nef in Ad5 naïve and Ad5 immune NHP. Additionally, NHP were immunized with an Ad5 [E1-, E2b-]-HIV-pol construct following two homologous administrations of Ad5 [E1-, E2b-]-SIV-gag/nef to determine if an immune response could be induced against a third antigen in the presence of vaccine induced Ad5 immunity. Positive CMI responses, as assessed by interferon-gamma (IFN-γ) secreting lymphocytes, were induced against all three antigens. These CMI responses increased over a course of multiple immunizations and the response profiles observed in Ad5 naïve and Ad5 immune NHP were similar. No influence of the major histocompatibility complex on CMI responses was observed. These data indicate that the new Ad5 [E1-, E2b-] platform based vaccine could be used for homologous vaccination regimes to induce robust CMI responses in the presence of Ad5 vector immunity., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The Nef protein is an essential factor for lentiviral pathogenesis in humans and other simians. Despite a multitude of functions attributed to this protein, the exact role of Nef in disease progression remains unclear. One of its most intriguing functions is the ability of Nef to enhance the infectivity of viral particles. In this review we will discuss current insights in the mechanism of this well-known, yet poorly understood Nef effect. We will elaborate on effects of Nef, on both virion biogenesis and the early stage of the cellular infection, that might be involved in infectivity enhancement. In addition, we provide an overview of different HIV-1 Nef domains important for optimal infectivity and briefly discuss some possible sources of the frequent discrepancies in the field. Hereby we aim to contribute to a better understanding of this highly conserved and therapeutically attractive Nef function.

Viruses like HIV and SIV escape from containment by CD8(+) T lymphocytes through generating mutations that interfere with epitope peptide:MHC class I binding. However, mutations in some viral epitopes are selected for that have no impact on this binding. We explored the mechanism underlying the evolution of such epitopes by studying CD8(+) T lymphocyte recognition of a dominant Nef epitope of SIVmac251 in infected Mamu-A*02(+) rhesus monkeys. Clonal analysis of the p199RY-specific CD8(+) T lymphocyte repertoire in these monkeys indicated that identical T cell clones were capable of recognizing wild-type (WT) and mutant epitope sequences. However, we found that the functional avidity of these CD8(+) T lymphocytes for the mutant peptide:Mamu-A*02 complex was diminished. Using surface plasmon resonance to measure the binding affinity of the p199RY-specific TCR repertoire for WT and mutant p199RY peptide:Mamu-A*02 monomeric complexes, we found that the mutant p199RY peptide:Mamu-A*02 complexes had a lower affinity for TCRs purified from CD8(+) T lymphocytes than did the WT p199RY peptide:Mamu-A*02 complexes. These studies demonstrated that differences in TCR affinity for peptide:MHC class I ligands can alter functional p199RY-specific CD8(+) T lymphocyte responses to mutated epitopes, decreasing the capacity of these cells to contain SIVmac251 replication.

HIV-1 and SIV Nef proteins downregulate cell surface CD4 and MHC class I (MHC-I) molecules of infected cells, which are necessary for efficient viral replication and pathogenicity. We previously reported that K144 in HIV-1 Nef is di-ubiquitinated, and K144R substitution impairs Nef-mediated CD4 downregulation. In this report, we extend the role of ubiquitination at this lysine residue from Nef-mediated CD4 downregulation to Nef-mediated MHC-I downregulation and from HIV Nef to SIV Nef. All HIV-1 Nef mutants that contain K144R substitution are inactive in MHC-I downregulation. Tested MHC-I alleles include HLA-ABC endogenously expressed and HLA-A2 exogenously expressed in Jurkat T cells. CD4 downregulation by SIV Nef involves K176 that aligns with K144 in HIV-1 Nef, as well as an N-terminal tyrosine motif Y28Y39 not present in HIV-1 Nef. Dual mutation at K176 and Y28Y39 completely impaired SIV Nef-mediated CD4 and MHC-I downregulation, whereas a single mutation at K176 or Y28Y39 did not. The involvement of tyrosine motif in SIV Nef-mediated CD4 and MHC-I downregulation prompted us to investigate a putative tyrosine motif (Y202Y/F203) in HIV-1 Nef that is conserved among HIV-1 species. Single mutation at the tyrosine motif Y202F203 in HIV-1 Nef (NA7) greatly impaired Nef-mediated CD4 downregulation, which is similar to what we observed previously with the single mutation at lysine K144. Thus, our study demonstrated that Nef-mediated receptor endocytosis involves the ubiquitination motif and tyrosine motif.

Lentiviral Nef proteins have multiple functions and are important for viral pathogenesis. Recently, Nef proteins from many simian immunodefiency viruses were shown to antagonize a cellular antiviral protein, named Tetherin, that blocks release of viral particles from the cell surface. However, the mechanism by which Nef antagonizes Tetherin is unknown. Here, using related Nef proteins that differ in their ability to antagonize Tetherin, we identify three amino-acids in the C-terminal domain of Nef that are critical specifically for its ability to antagonize Tetherin. Additionally, divergent Nef proteins bind to the AP-2 clathrin adaptor complex, and we show that residues important for this interaction are required for Tetherin antagonism, downregulation of Tetherin from the cell surface and removal of Tetherin from sites of particle assembly. Accordingly, depletion of AP-2 using RNA interference impairs the ability of Nef to antagonize Tetherin, demonstrating that AP-2 recruitment is required for Nef proteins to counteract this antiviral protein.

Background: The non-pathogenic course of SIV infection in its natural host is characterized by robust viral replication in the absence of chronic immune activation and T cell proliferation. In contrast, acutely lethal enteropathic SIVsmm strain PBj induces a strong immune activation and causes a severe acute and lethal disease in pig-tailed macaques after cross-species transmission. One important pathogenicity factor of the PBj virus is the PBj-Nef protein, which contains a conserved diacidic motif and, unusually, an immunoreceptor tyrosine-based activation motif (ITAM)., Results: Mutation of the diacidic motif in the Nef protein of the SIVsmmPBj abolishes the acute phenotype of this virus. In vitro, wild-type and mutant PBj (PBj-Nef202/203GG) viruses replicated to similar levels in macaque PBMCs, but PBj-Nef202/203GG no longer triggers ERK mitogen-activated protein (MAP) kinase pathway including an alteration of a Nef-associated Raf-1/ERK-2 multiprotein signaling complex. Moreover, stimulation of IL-2 and down-modulation of CD4 and CD28 were impaired in the mutant virus. Pig-tailed macaques infected with PBj-Nef202/203GG did not show enteropathic complications and lethality as observed with wild-type PBj virus, despite efficient replication of both viruses in vivo. Furthermore, PBj-Nef202/203GG infected animals revealed reduced T-cell activation in periphery lymphoid organs and no detectable induction of IL-2 and IL-6., Conclusions: In sum, we report here that mutation of the diacidic motif in the PBj-Nef protein abolishes disease progression in pig-tailed macaques despite efficient replication. These data suggest that alterations in the ability of a lentivirus to promote T cell activation and proliferation can have a dramatic impact on its pathogenic potential.

There are several diseases for which gene transfer therapy to the cerebellum might be practicable. In these studies, we used recombinant Tag-deleted SV40-derived vectors (rSV40s) to study gene delivery targeting the cerebellum. These vectors transduce neurons and microglia very effectively in vitro and in vivo, and so we tested them to evaluate gene transfer to the cerebellum in vivo. Using a rSV40 vector carrying human immunodeficiency virus (HIV)-Nef with a C-terminal FLAG epitope, we characterized the distribution, duration, and cell types transduced. Rats received test and control vectors by stereotaxic injection into the cerebellum. Transgene expression was assessed 1, 2, and 4 weeks later by immunostaining of serial brain sections. FLAG epitope-expressing cells were seen, at all times after vector administration, principally detected in the Purkinje cells of the cerebellum, identified as immunopositive for calbindin. Occasional microglial cells were tranduced; transgene expression was not detected in astrocytes or oligodendrocytes. No inflammatory or other reaction was detected at any time. Thus, SV40-derived vectors can deliver effective, safe, and durable transgene expression to the cerebellum.

Pathogenic simian-human immunodeficiency viruses (SHIV) contain HIV-1 Vpu and SIV Nef, both shown to counteract BST-2 (HM1.24; CD317; tetherin) inhibition of virus release in a species-specific manner. We show that human and pig-tailed BST-2 (ptBST-2) restrict SHIV. We found that sequential "humanization" of the transmembrane domain (TMD) of the pig-tailed BST-2 (ptBST-2) protein resulted in a fluctuation in sensitivity to HIV-1 Vpu. Our results also show that the length of the TMD in human and ptBST-2 proteins is important for BST-2 restriction and susceptibility to Vpu. Taken together, our results emphasize the importance of tertiary structure in BST-2 antagonism and suggests that the HIV-1 Vpu transmembrane domain may have additional functions in vivo unrelated to BST-2 antagonism., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Tetherin/BST-2 is a host-encoded protein that restricts a wide diversity of viruses at the stage of virion release. However, viruses have evolved antagonists of Tetherin, including the Vpu and Nef proteins of primate lentiviruses. Like other host genes subject to viral antagonism, primate Tetherin genes have evolved under positive selection. We show here that viral antagonists acting at three independent sites of selection have driven the evolution of Tetherin, with the strongest selective pressure on the cytoplasmic tail domain. Human Tetherin is unique among the Tetherins of simian primates in that it has a 5-amino-acid deletion that results in the loss of the residue under the strongest positive selection. We show that this residue at amino acid 17 is the site of the functional interaction of Tetherin with Nef, since single amino acid substitutions at this single position can determine the susceptibility of Tetherin to Nef antagonism. While the simian immunodeficiency viruses SIVcpz and SIVgor are able to antagonize their hosts' Tetherin with Nef, human immunodeficiency virus type 1 (HIV-1) Vpu has evolved to counteract Tetherin in humans. We mapped the adaptations in the N-terminal transmembrane domain of Vpu that allow it to counteract human Tetherin. Our combined evolutionary and functional studies have allowed us to reconstruct the host-pathogen interactions that have shaped Tetherin and its lentivirus-encoded antagonists.

Previous studies demonstrated that the nef gene is a critical determinant of the pathogenicity of simian immunodeficiency virus (SIV) in macaques. In the present study, we evaluated the effect of a spontaneous frameshift mutation in the C-terminus of the nef gene of the minimally pathogenic SIVsmH4i clone. This clone exhibited a single nucleotide deletion in the nef gene relative to pathogenic SIV clones that resulted in a frameshift and addition of 46 amino acids to the C-terminus of Nef. We generated a corrected version of this clone, SIVsmH4i Nef+ that restored Nef protein expression. Inoculation of macaques with SIVsmH4i resulted in delayed and low levels of peak viremia. This contrasted with improved kinetics and robust peak viremia in macaques inoculated with the corrected version. Despite the restoration of in vivo replication ability, neither clone resulted in memory CD4+ T cell loss or disease in a period of two years., (Published by Elsevier Inc.)

SIVsmmFGb is a lentivirus swarm that induces neuropathology in over 90% of infected pigtailed macaques and reliably models central nervous system HIV infection in people. We have previously studied SIVsmmFGb genetic diversity and compartmentalization during acute infection, but little is understood about diversity and intertissue compartmentalization during chronic infection. Tissue-specific pressure appeared to affect the diversity of Nef sequences between tissues, but changes to the Env V1 region and Int diversity were similar across all tissues. At 2 months postinfection, compartmentalization of the SIVsmmFGb env V1 region, nef, and int was noted between different brain regions and between brain regions and lymph nodes. Convergent evolution of the nef and env V1 region, and divergent evolution of int, was noted between compartments and all genes demonstrated intratissue temporal segregation. For the env V1 region and nef, temporal segregation was stronger in the brain regions than the periphery, but little difference between tissues was noted for int. Positive selection of the env V1 region appeared in most tissues at 2 months postinfection, whereas nef and int faced negative selection in all tissues. Positive selection of the env V1 region sequences increased in some brain regions over time. SIVsmmFGb nef and int sequences each saw increased negative selection in brain regions, and one lymph node, over the course of infection. Functional differences between tissue compartments decreased over time for int and env V1 region sequences, but increased for nef sequences.

Background: Försters resonance energy transfer (FRET) microscopy is widely used for the analysis of protein interactions in intact cells. However, FRET microscopy is technically challenging and does not allow assessing interactions in large cell numbers. To overcome these limitations we developed a flow cytometry-based FRET assay and analysed interactions of human and simian immunodeficiency virus (HIV and SIV) Nef and Vpu proteins with cellular factors, as well as HIV Rev multimer-formation., Results: Amongst others, we characterize the interaction of Vpu with CD317 (also termed Bst-2 or tetherin), a host restriction factor that inhibits HIV release from infected cells and demonstrate that the direct binding of both is mediated by the Vpu membrane-spanning region. Furthermore, we adapted our assay to allow the identification of novel protein interaction partners in a high-throughput format., Conclusion: The presented combination of FRET and FACS offers the precious possibility to discover and define protein interactions in living cells and is expected to contribute to the identification of novel therapeutic targets for treatment of human diseases.

Infection with Simian Immunodeficiency Virus (SIV) leads to high viral loads and progression to Simian AIDS (SAIDS) in rhesus macaques. The viral accessory protein Nef is required for this phenotype in monkeys as well as in HIV-infected humans. Previously, we determined that HIVNef binds HIVGagPol and Alix for optimal viral replication in cells. In this study, we demonstrated that these interactions could correlate with high viral loads leading to SAIDS in the infected host. By infecting rhesus macaques with a mutant SIV(mac239), where sequences in the nef gene that are required for these interactions were mutated, we observed robust viral replication and disease in two out of four monkeys, where they reverted to the wild type genotype and phenotype. These two rhesus macaques also died of SAIDS. Two other monkeys did not progress to disease and continued to harbor mutant nef sequences. We conclude that interactions between Nef, GagPol and Alix contribute to optimal viral replication and progression to disease in the infected host.

Many aspects of HIV-1 pathogenesis are affected by Nef protein activity, and efforts have been made to study variation in the nef gene and how that variation relates to disease outcome. We studied the genetic diversity of the nef gene in distinct clones obtained from the same patient (intrahost) and in sequences obtained from different hosts (interhost). The set of sequences analyzed was obtained from HIV-1-infected Brazilian children and contained 112 clones from 25 children (intrahost samples), as well as 55 sequences from epidemiologically unlinked children (interhost samples). We found extensive site polymorphisms and amino acid length variations, mainly in the amino terminal region of the nef gene, between the myristoylation motif (MGxxxS) and the MHC-1 downregulation motif (Rxx). Analysis of the sequences deposited in the Los Alamos HIV sequences database ( www.hiv.lanl.gov ) indicated that the most frequent motif at the MHC-1 downregulation site in the subtype B strain is R(86%)A(64%)E(82%) (n = 1040) and R(78%)T(74%)E(56%) in the subtype C strain (n = 549). Conversely, the Brazilian subtype B isolates presented the motif R(81%)T(62%)E(67%) at this site (n = 64). A detailed analysis of selective pressures identified a concentration of codons under strong positive selection in the amino terminal region of the nef gene. We also determined that different sites are under positive selection in the subtype B and subtype C viruses. The amino acid composition in the MHC-1 downregulation motif of the nef gene in our sequences may indicate a distinct adaptive pattern of HIV-1 subtype B to the Brazilian host population.

Comparison of mutant killer cell Ig-like receptor (KIR) 3DL1*015 substituted at natural positions of variation showed that tryptophan/leucine dimorphism at position 283 uniquely changes receptor conformation and can strongly influence binding of the A24nef tetramer. Dimorphic motifs at positions 2, 47, and 54 in D0 and 182 and 283 in D1+D2 distinguish the two 3DL1 lineages, typified by 3DL1*005 and 3DL1*015. The interlineage recombinant, KIR3DL1*001, combines D0 of 3DL1*005 with D1+D2 of 3DL1*015 and binds A24nef more strongly than either parent. In contrast, the reciprocal recombinant with D0 from 3DL1*015 and D1+D2 from 3DL1*005 cannot bind A24nef. Thus, D0 polymorphism directly affects the avidity of the KIR3DL1 ligand binding site. From these observations, multiple sequence alignment, and homology modeling, we constructed structural models for KIR3DL1 and its complex with A24nef. In these models, D0, D1, and D2 come together to form a binding surface for A24nef, which is contacted by all three Ig-like domains. A central pocket binds arginine 83, the only Bw4 motif residue essential for KIR3DL1 interaction, similar to the binding of lysine 80 in HLA-C by KIR2DL1. Central to this interaction is a salt bridge between arginine 83 of Bw4 and glutamate 282 of 3DL1, which juxtaposes the functionally influential dimorphism at position 283. Further 3DL1 mutants were tested and shown to have A24nef-binding properties consistent with the models. A24nef was not bound by KIR3DS1, the activating counterpart of KIR3DL1. Moreover, introducing any one of three residues specific to KIR3DS1, serine 163, arginine 166, or leucine 199, into 3DL1*015, abrogated A24nef binding.

Microglia are macrophage-like cells in the CNS. As macrophages, activated microglia remove potentially deleterious debris and promote tissue repair. However, they can release potentially cytotoxic substances in vitro. So-called fully activated microglia, observed at the injury site in many neurodegenerative conditions, are neurotoxic. This suggests that some factor(s) may contribute to change microglial phenotype from protective to toxic, but details are not clear. Recently, we generated HIV-derived Nef protein-transduced microglia. They increase the potential to produce O(-)(2) and MPO-like peroxidase activity, resulting in neurotoxicity. Therefore, the target protein(s) of Nef might be involved in the control of microglial neurotoxicity.

Objective: To study the specific amino acid variation in Nef that may be related to disease progression after infection with HIV-1 subtype B, a predominant strain circulating in China, and to determine whether changes in Nef secondary structure may influence different stages of AIDS development based on the concept that the Nef gene of HIV infection dramatically alter the severity of viral infection and virus replication and disease progression, and that long-term non-progressors (LTNP) of HIV infection are commonly associated with either a deletion of the Nef gene or the defective Nef alleles., Methods: The study subjects were divided into LTNP1(n=14), LTNP2 (n=16) and slow progressor (SP, n=19) groups for mutational analysis of the Nef sequence. The data were obtained by using Bioedit, MEGA, Anthewin and SAS software., Results: Residues in Nef TA(48/49) and K151 occurred more frequently in the LTNP group while AA(48/49) was more frequently observed in the SP group. Of the differences observed in the secondary structure comparison using Nef consensus sequences of these three groups, one was roughly corresponding to the Nef(48/49) mutation site., Conclusion: TA(48/49), K(151), and AA(48/49) in the Nef gene might be associated with the different stages of HIV infection, and there may be a link between the Nef secondary structure and the progression of HIV-1 infection.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef downregulates major histocompatibility complex class I (MHC-I) from the cell surface. It has been proposed that the direct interaction of the acidic cluster (AC) of Nef, (62)EEEE(65), with the furin binding region (fbr) of PACS-1 is crucial for this Nef function. Contrary to this proposal, evidence is presented here that the four glutamates in Nef do not functionally engage the PACS-1 fbr. (i) The binding of Nef to the PACS-1 fbr in vitro is much weaker than the binding of the canonical furin AC to the PACS-1 fbr. (ii) The mutation of two of the four glutamates in Nef's AC to alanines does not alter Nef's ability to downregulate MHC-I, and triply mutated Nefs exhibit 50% activity. (iii) The introduction of lysine into the AC has little effect on Nef function. (iv) The mutation of all four glutamates to alanine does debilitate Nef MHC-I downregulation, but this quadruple mutation also impairs the ability of Nef to regulate p21-activated protein kinase and enhance viral particle infectivity. (v) The replacement of the Nef AC with the bona fide AC from furin results in the loss of the expected regulatory properties of the furin AC. (vi) The insertion of the conformation-disrupting amino acid proline into the Nef AC does not disrupt MHC-I downregulation. Our results are consistent with an alternative model in which (62)EEEE(65) plays a stabilizing role in the formation of a ternary complex between Nef, the MHC-I cytoplasmic domain, and AP-1.

Despite reports of viral genetic defects in persons who control human immunodeficiency virus type 1 (HIV-1) in the absence of antiviral therapy, the extent to which such defects contribute to the long-term containment of viremia is not known. Most previous studies examining for such defects have involved small numbers of subjects, primarily focused on subjects expressing HLA-B57, or have examined single viral genes, and they have focused on cellular proviral DNA rather than plasma viral RNA sequences. Here, we attempted viral sequencing from 95 HIV-1 elite controllers (EC) who maintained plasma viral loads of <50 RNA copies/ml in the absence of therapy, the majority of whom did not express HLA-B57. HIV-1 gene fragments were obtained from 94% (89/95) of the EC, and plasma viral sequences were obtained from 78% (61/78), the latter indicating the presence of replicating virus in the majority of EC. Of 63 persons for whom nef was sequenced, only three cases of nef deletions were identified, and gross genetic defects were rarely observed in other HIV-1 coding genes. In a codon-by-codon comparison between EC and persons with progressive infection, correcting for HLA bias and coevolving secondary mutations, a significant difference was observed at only three codons in Gag, all three of which represented the historic population consensus amino acid at the time of infection. These results indicate that the spontaneous control of HIV replication is not attributable to shared viral genetic defects or shared viral polymorphisms.

More than a decade ago it was established that intact nef genes are critical for efficient viral persistence and greatly accelerate disease progression in SIVmac-infected rhesus macaques and in HIV-1-infected humans. Subsequent studies established a striking number of Nef functions that evidently contribute to the maintenance of high viral loads associated with the development of immunodeficiency in the 'evolutionary-recent' human and the experimental macaque hosts. Recent data show that many Nef activities are conserved across different lineages of HIV and SIV. However, some differences also exist. For example, Nef alleles from most SIVs that do not cause disease in their natural monkey hosts, but not those of HIV-1 and its simian precursors, down-modulate TCR-CD3 to suppress T cell activation and programmed death. This evolutionary loss of a specific Nef function may contribute to the high virulence of HIV-1 in humans.

The levels of accumulation of recombinant vaccines in transgenic plants are protein specific and strongly influenced by the subcellular compartment of destination. The human immunodeficiency virus protein Nef (negative factor), a promising target for the development of an antiviral vaccine, is a cytosolic protein that accumulates to low levels in transgenic tobacco and is even more unstable when introduced into the secretory pathway, probably because of folding defects in the non-cytosolic environment. To improve Nef accumulation, a new strategy was developed to anchor the molecule to the cytosolic face of the endoplasmic reticulum (ER) membrane. For this purpose, the Nef sequence was fused to the C-terminal domain of mammalian ER cytochrome b5, a long-lived, tail-anchored (TA) protein. This consistently increased Nef accumulation by more than threefold in many independent transgenic tobacco plants. Real-time polymerase chain reaction of mRNA levels and protein pulse-chase analysis indicated that the increase was not caused by higher transcript levels but by enhanced protein stability. Subcellular fractionation and immunocytochemistry indicated that Nef-TA accumulated on the ER membrane. Over-expression of mammalian or plant ER cytochrome b5 caused the formation of stacked membrane structures, as observed previously in similar experiments performed in mammalian cells; however, Nef-TA did not alter membrane organization in tobacco cells. Finally, Nef could be removed in vitro by its tail-anchor, taking advantage of an engineered thrombin cleavage site. These results open up the way to use tail-anchors to improve foreign protein stability in the plant cytosol without perturbing cellular functions.

Nef is a HIV-1 accessory protein critical for the replication of the virus and the development of AIDS. The major pathological activity of Nef is the down-regulation of CD4, the primary receptor of HIV-1 infection. The mechanism underlying Nef-mediated CD4 endocytosis and degradation remains incompletely understood. Since protein ubiquitination is the predominant sorting signal in receptor endocytosis, we investigated whether Nef is ubiquitinated. The in vivo ubiquitination assay showed that both HIV-1 and SIV Nef proteins expressed in Jurkat T cells and 293T cells were multiple ubiquitinated by ubiquitin-His. The lysine-free HIV-1 Nef mutant (Delta10K) generated by replacing all 10 lysines with arginines was not ubiquitinated and the major ubiquitin-His attachment sites in HIV-1 Nef were determined to be lysine 144 (di-ubiquitinated) and lysine 204 (mono-ubiquitinated). Lysine-free HIV-1 Nef was completely inactive in Nef-mediated CD4 down-regulation, so was the Nef mutant with a single arginine substitution at K144 but not at K204. A mutant HIV-1 provirion NL4-3 with a single arginine substitution in Nef at K144 was also inactive in Nef-mediated CD4 down-regulation. Lysine-free Nef mutant reintroduced with lysine 144 (DeltaK10 + K144) was shown active in CD4 down-regulation. These data suggest that ubiquitination of Nef, particularly diubiquitination of the lysine 144, is necessary for Nef-mediated CD4 down-regulation.

Infection of primates by HIV-1 and SIV induces multiple hematological abnormalities of central hematopoietic origin. Although these defects greatly contribute to the pathophysiology of HIV-1 infection, the molecular basis for altered BM function remains unknown. Here we show that when cynomolgus macaques were infected with SIV, the multipotent potential of their hematopoietic progenitor cells was lost, and this correlated with downregulation of STAT5A and STAT5B expression. However, forced expression of STAT5B entirely rescued the multipotent potential of the hematopoietic progenitor cells. In addition, an accessory viral protein required for efficient SIV and HIV replication and pathogenicity, "Negative factor" (Nef), was essential for SIV-mediated impairment of the multipotent potential of hematopoietic progenitors ex vivo and in vivo. This newly uncovered property of Nef was both conserved between HIV-1 and SIV strains and entirely dependent upon the presence of PPARgamma in targeted cells. Further, PPARgamma agonists mimicked Nef activity by inhibiting STAT5A and STAT5B expression and hampering the functionality of hematopoietic progenitors both ex vivo and in vivo. These findings have extended the role of Nef in the pathogenicity of HIV-1 and SIV and reveal a pivotal role for the PPARgamma/STAT5 pathway in the regulation of early hematopoiesis. This study may provide a basis for investigating the potential therapeutic benefits of PPARgamma antagonists in both patients with AIDS and individuals with hematopoietic disorders.

An effective vaccine against HIV-1 is generally considered the best hope for controlling the raging AIDS pandemic. As a part of our AIDS vaccine development effort, we constructed a dual-promoter plasmid capable of high-level expression of 2 independent transgenes. HIV-1 gag, pol, env, nef, and tat from a primary subtype C/B' CCR5-tropic HIV-1 were "codon" optimized, modified to eliminate known functional activity, and assembled using an overlapping polymerase chain reaction into 2 plasmids: ADVAX-I (containing env and gag) and ADVAX-II (containing pol and nef-tat). These 2 dual-promoter candidate vaccines showed levels of HIV-1 gene expression comparable to those observed with single-gene plasmids in vitro. Importantly, immunization of mice with these vaccine constructs resulted in dose-dependent multigenic CD4 and CD8 T-cell responses equivalent to those provided by vaccination with single-gene plasmids. With input from the US Food and Drug Administration, ADVAX-I and ADVAX-II have since been combined as a single candidate DNA vaccine, ADVAX. A phase 1 clinical trial of this product has been successfully completed, and its use in prime-boost studies is now underway.

The rapid spread of HIV-1 underscores the urgent need to develop an effective vaccine. Using modified vaccinia Ankara (MVA) as a vector, we designed and constructed a multigenic candidate vaccine against a recombinant C/B' subtype of HIV-1 that is dominant in southwest China. Five HIV-1 genes (gag, pol, DeltaV2env, tat, and nef) were introduced into 2 separate regions of the MVA genome using modified single- and dual-promoter insertion vectors. Recombinant MVA was selected by immunofluorescence double-staining and foci purification. The end product is a single recombinant MVA, termed ADMVA, that expresses HIV-1 DeltaV2Env and fusion proteins Gag-Pol and Nef-Tat. By in vitro analyses, all expected HIV-1 proteins were expressed in infected chicken embryo fibroblasts and various human cell lines. Additionally, 2 sequential intramuscular injections of 10(6) 50% tissue infectious culture dose (TCID50) of ADMVA into BALB/c and B6 x B10 mice elicited broad cell-mediated immune responses against all 5 viral proteins as determined by interferon-gamma enzyme immunospot assays. The number of spot-forming cells was in the range of 200 to 800 per million splenocytes, and both CD4 and CD8 T-cell responses were detected. Moreover, high serum titers (>1:20,000) of antibodies against HIV-1 gp120 were also elicited. The magnitude of immune responses correlated with the dose of ADMVA, and the vaccine caused no overt adverse consequences, up to 10(7) TCID50 per injection. ADMVA has since been advanced into clinical trials. A phase 1 study has been completed, and a prime-boost with ADVAX (see accompanying article) is now underway.