Substitution of HIV Type 1 Nef with HTLV-1 p12

Human retroviruses, such as HTLV-1 and HIV-1, encode accessory proteins, which regulate viral pathogenesis. The p12 protein of HTLV-1 is encoded from the pX-I open reading frame, and is critical for efficient virus replication in rabbits. Although dispensable for infection, replication, and immortalization of activated lymphocytes in culture, p12 expression is important for infection of quiescent lymphocytes. Similar to HTLV-1 p12, Nef is important for virus infectivity in SIV animal models. We questioned whether p12 could replace Nef in HIV-1, and reconstitute virus replication in culture. We found that p12 could complement for effects of Nef on HIV-1 infection of Magi-CCR5 cells or macrophages.     

Partial "repair" of defective NEF genes in a long-term nonprogressor with human immunodeficiency virus type 1 infection

A 36-bp deletion close to the 5' end of NEF that impaired Nef function was found in a long-term nonprogressor with human immunodeficiency virus type 1 (HIV-1) infection. Forms containing an adjacent duplication of 33 bp were also frequently observed. The duplication showed no homology to the deleted region but restored the overall length of the first variable loop of Nef. NEF alleles carrying the duplication were active in class I major histocompatibility complex (MHC-I) down-modulation and enhancement of virus infectivity. However, they showed little activity in CD4 down-regulation and were unable to stimulate viral replication in human peripheral blood mononuclear cells. Our study indicates that the enhancement of virion infectivity and the stimulation of HIV-1 replication in lymphocytes are distinct functions of Nef. Our findings also illustrate the capacity for repair of attenuating deletions in HIV-1 infection and suggest that a selective pressure for Nef-mediated MHC-I down-modulation and/or enhancement of virion infectivity exists.     

Nef and TNFalpha are coplayers that favor HIV-1 replication in monocytic cells and primary macrophages

The human immunodeficiency virus (HIV) Nef protein is myristoylated and plays a critical role in AIDS pathogenesis by enhancing viral replication, survival of the virus within infected cells and by facilitating its spread in vivo. We observed that, in the promonocytic cell line U937, myristoylated exogenous Nef protein activates NF-kappaB and AP-1, whereas unmyristoylated exogenous Nef protein does not. Using methyl-beta-cyclodextrin (MbetaC) treatment, we observed that the activation of NF-kappaB and AP-1 by exogenous Nef protein is mediated primarily via lipid rafts both in U937 cells and in primary human macrophages. In agreement with this observation, exogenous Nef protein colocalized with GM1 ganglioside, a major component of lipid rafts, in U937 cells as detected by confocal microscopy. Since tumor necrosis factor alpha (TNFalpha) activates NF-kappaB and AP-1, we investigated the role of exogenous Nef protein in TNFalpha-stimulated U937 cells and primary macrophages. We observed that exogenous Nef and TNFalpha synergistically activate NF-kappaB and AP-1 in U937 cells and primary macrophages resulting in enhanced stimulation of the HIV-1 long terminal repeat (LTR), and subsequently in enhanced viral replication in both chronically infected promonocytic U1 cells and acutely HIV-1-infected primary macrophages. Both enhanced LTR stimulation and viral replication following treatment with exogenous Nef and TNFalpha were mediated via lipid rafts. Therefore, our results indicate that exogenous Nef protein and enhanced TNFalpha production detected in HIV-infected subjects could synergize to fuel the progression of the disease via lipid raft-dependent stimulation of the HIV-1 provirus present in such cellular reservoirs as mononuclear phagocytes.     

HIV-1 Nef interferes with M-CSF receptor signaling through Hck activation and inhibits M-CSF bioactivities

HIV-1 Nef protein is a major determinant of the pathogenicity of the virus. It has been shown that Nef activates Hck, a member of Src family kinase, in monocytes/macrophages and that the interaction is critical for AIDS-like disease progression in a mouse model. However, it was unclear how the molecular interaction in monocytes/macrophages leads to disease progression. Here, we show for the first time that Nef interferes with the macrophage colony-stimulating factor (M-CSF)/M-CSF receptor signal pathway. In this study, we introduced a conditionally active Nef into myeloid leukemia TF-1-fms cells and analyzed their responsiveness to M-CSF. We found that Nef-activated Hck constitutively associated with the M-CSF receptor complex. The formation of the molecular complex should occur under physiologic conditions, that is, on M-CSF stimulation. Because of aberrant molecular association, the tyrosine-phosphorylation/activation of the receptor in response to M-CSF was markedly diminished in Nef-active cells. Consequently, Nef activation caused the inhibition of M-CSF-mediated proliferation of TF-1-fms cells and macrophage differentiation of the cells induced by M-CSF and 12-O-tetradecanoylphorbol 13-acetate. These results indicate that HIV-1 Nef interferes with M-CSF receptor signaling through Hck activation and thereby inhibits M-CSF functions in monocytes/macrophages.     

Interaction between Hck and HIV-1 Nef negatively regulates cell surface expression of M-CSF receptor

Nef is a multifunctional pathogenetic protein of HIV-1, the interaction of which with Hck, a Src tyrosine kinase highly expressed in macrophages, has been shown to be responsible for the development of AIDS. However, how the Nef-Hck interaction leads to the functional aberration of macrophages is poorly understood. We recently showed that Nef markedly inhibited the activity of macrophage colony-stimulating factor (M-CSF), a primary cytokine for macrophages. Here, we show that the inhibitory effect of Nef is due to the Hck-dependent down-regulation of the cell surface expression of M-CSF receptor Fms. In the presence of Hck, Nef induced the accumulation of an immature under-N-glycosylated Fms at the Golgi, thereby down-regulating Fms. The activation of Hck by the direct interaction with Nef was indispensable for the down-regulation. Unexpectedly, the accumulation of the active Hck at the Golgi where Nef prelocalized was likely to be another critical determinant of the function of Nef, because the expression of the constitutive-active forms of Hck alone did not fully down-regulate Fms. These results suggest that Nef perturbs the intracellular maturation and the trafficking of nascent Fms, through a unique mechanism that required both the activation of Hck and the aberrant spatial regulation of the active Hck.     

Lentivirus vectors expressing short hairpin RNAs against the U3-overlapping region of HIV nef inhibit HIV replication and infectivity in primary macrophages

Although successful attempts to inhibit HIV-1 replication in T cells using RNAi have been reported, the effect of HIV-specific RNAi on macrophages is not well known. Macrophages are key targets for anti-HIV-1 therapy because they are able to survive long after the initial infection with HIV and can spread the virus to T cells. In this study, we identified a putative RNAi target of HIV, consisting of the portion of the nef gene overlapping the U3 region (Nef366), and generated a lentivirus-based short hairpin RNA (shRNA) expression vector (Lenti shNef366). We show that Lenti shNef366 inhibits (1) HIV-1 replication in a monocytic cell line and in primary monocyte-derived macrophages (MDMs), (2) reactivation of latent HIV-1 infection, and (3) the production of secondary HIV-1 from MDMs harboring a genomic copy of Nef366. Moreover, we found that the up-regulated production of macrophage inflammatory protein 1beta (MIP-1beta), but not MIP-1alpha, in MDMs by Nef expression was considerably suppressed by Lenti shNef366, which suggests that HIV-1 dissemination to T cells through its interaction with HIV-1-infected MDMs can also be controlled by Lenti shNef366. Thus, lentivirus-mediated shRNA expression targeting the U3-overlapping region of HIV nef represents a feasible approach to genetic vaccine therapy for HIV-1.     

HIV-1 Nef down-regulates the hemochromatosis protein HFE, manipulating cellular iron homeostasis

The multifunctional Nef protein of HIV-1 is important for the progression to AIDS. One action of Nef is to down-regulate surface MHC I molecules, helping infected cells to evade immunity. We found that Nef also down-regulates the macrophage-expressed MHC 1b protein HFE, which regulates iron homeostasis and is mutated in the iron-overloading disorder hemochromatosis. In model cell lines, Nef reroutes HFE to a perinuclear structure that overlaps the trans-Golgi network, causing a 90% reduction of surface HFE. This activity requires a Src-kinase-binding proline-rich domain of Nef and a conserved tyrosine-based motif in the cytoplasmic tail of HFE. HIV-1 infection of ex vivo macrophages similarly down-regulates naturally expressed surface HFE in a Nef-dependent manner. The effect of Nef expression on cellular iron was explored; iron and ferritin accumulation were increased in HIV-1-infected ex vivo macrophages expressing wild-type HFE, but this effect was lost with Nef-deleted HIV-1 or when infecting macrophages from hemochromatosis patients expressing mutated HFE. The iron accumulation in HIV-1-infected HFE-expressing macrophages was paralleled by an increase in cellular HIV-1-gag expression. We conclude that, through Nef and HFE, HIV-1 directly regulates cellular iron metabolism, possibly benefiting viral growth.     

Role of the CD4 down-modulation activity of Nef in HIV-1 infectivity

The regulatory Nef protein of HIV-1/2 and SIV is required for high viral replication and disease progression, thus represents a very attractive therapeutic target. Because of the multi-functional nature of the Nef protein, it is unclear which of the several Nef activities are most crucial in vivo for the outcome of viral infection. Some findings indicate that the CD4 down-regulation activity of Nef is critical for viral infectivity as well as for progression to immunodeficiency. On the other hand, more recent evidences suggest that CD4 targeting and stimulation of infectivity are two separate functions of Nef. This controversial issue will be discussed here in the light of the latest findings.     

Co-infection with opportunistic pathogens promotes human immunodeficiency virus type 1 infection in macrophages

Human immunodeficiency virus type 1 (HIV-1) infection is dependent on susceptible host cells that express both CD4 and chemokine co-receptors. The co-receptor CCR5 is associated with primary infection by macrophage-tropic virus isolates, whereas CXCR4 is commonly associated with T cell- and dual-tropic viruses. Once infected, lymphocytes and macrophages may replicate HIV-1 or harbor latent virus, depending on environmental factors and cellular activation. Immune activation is often associated with viremia, which is consistent with enhanced infection and viral replication in activated cells harboring virus. In this regard, opportunistic infections activate the immune system with the detrimental sequelae of enhanced viral replication and viremia. Under these conditions, viral expansion extends beyond T cells to tissue macrophages, many of which are co-infected with opportunistic pathogens. The opportunistic infections promote macrophage susceptibility to HIV-1 through cytokine modulation and altered chemokine co-receptors, potential targets for intervention.     

HIV-1 Nef compensates for disorganization of the immunological synapse by inducing trans-Golgi network-associated Lck signaling

The Nef protein of HIV-1 facilitates viral replication and disease progression in vivo. Nef disturbs the organization of immunological synapses between infected CD4(+) T lymphocytes and antigen-presenting B-lymphocytes to interfere with TCR proximal signaling. Paradoxically, Nef enhances distal TCR signaling in infected CD4(+) T lymphocytes, an effect thought to be involved in its role in AIDS pathogenesis. Using quantitative confocal microscopy and cell fractionation of Nef-expressing cells and HIV-1-infected primary human T lymphocytes, we found that Nef induces intracellular compartmentalization of TCR signaling to adjust TCR responses to antigenic stimulation. Nef reroutes kinase-active pools of the TCR signaling master switch Lck away from the plasma membrane (PM) to the trans-Golgi network (TGN), thereby preventing the recruitment of active Lck to the immunological synapse after TCR engagement and limiting signal initiation at the PM. Instead, Nef triggers Lck-dependent activation of TGN-associated Ras-Erk signaling to promote the production of the T lymphocyte survival factor IL-2 and to enhance virus spread. Overexpression of the Lck PM transporter Unc119 restores Nef-induced subversions of Lck trafficking and TCR signaling. Nef therefore hijacks Lck sorting to selectively activate TGN-associated arms of compartmentalized TCR signaling. By tailoring T-lymphocyte responses to antigenic stimulation, Nef optimizes the environment for HIV-1 replication.     

nef-deleted HIV-1 inhibits phagocytosis by monocyte-derived macrophages in vitro but not by peripheral blood monocytes in vivo

OBJECTIVE: HIV-1 infection impairs a number of macrophage effector functions, but the mechanism is unknown. We studied the role of HIV-1 Nef in modulating phagocytosis by human monocytes and monocyte-derived macrophages (MDM). DESIGN AND METHODS: Using a flow cytometric assay, phagocytosis of Mycobacterium avium complex (MAC) by monocytes in whole blood of Sydney Blood Bank Cohort (SBBC) members infected with a nef-deleted (Delta nef) strain of HIV-1 was compared with that of monocytes from uninfected or wild-type (WT) HIV-infected subjects. The specific impact of Nef on phagocytosis by MDM was determined by either infecting cells in vitro with Delta nef strains of HIV-1 or electroporating Nef into uninfected MDM. RESULTS: MAC phagocytic capacity of monocytes from SBBC members was equivalent to that of cells from uninfected individuals (P = 0.81); it was greater than that of cells from individuals infected with WT HIV-1 (P < 0.0001), irrespective of CD4 counts and HIV viral load. In contrast, in vitro infection of MDM with either Delta nef or WT strains of HIV-1 resulted in similar levels of HIV replication and equivalent impairment of phagocytosis via Fc gamma and complement receptors. Electroporation of Nef into MDM did not alter phagocytic capacity. CONCLUSIONS: This study provides evidence demonstrating the complex indirect effect of Nef on phagocytosis by peripheral blood monocytes (infrequently infected with HIV-1) in vivo. Conversely, the fact that MDM infected with either Delta nef or WT HIV-1 in vitro (high multiplicity of infection) show comparably impaired phagocytosis, indicates that HIV-1 infection of macrophages can directly impair function, independent of Nef.     

Nuclear localization of HIV type 1 Vif isolated from a long-term asymptomatic individual and potential role in virus attenuation

Recent reports have determined that HIV-1 Vif counteracts an innate antiviral cellular factor, Apobec3G. However, the function of Vif during HIV-1 pathogenesis remains poorly understood. To gain a better understanding of Vif function, the viral isolate from an HIV-1-infected long-term nonprogressor (LTNP) that displayed a Vif-mutant replication phenotype was studied. This LTNP has been infected since before 1983 and has no HIV-related disease in the absence of antiretroviral therapy. From separate samples, obtained on more than one study visit, virus grew in cocultures of LTNP cells with Vif-complementing T cell lines, but not the parental T cell lines. An unusual amino acid motif (KKRK) was found in the Vif sequence at positions 90 to 93. Since this motif commonly functions as a nuclear localization sequence, experiments were performed to determine the ability of this KKRK motif to mediate nuclear localization of Vif. Wild-type Vif displayed a predominantly cytoplasmic distribution. In contrast, the KKRK Vif showed a predominantly nuclear localization. The effect of the KKRK mutation on virus production and infectivity was also studied. The KKRK motif that mislocalizes Vif to the nucleus also reduces viral replication and infectivity in nonpermissive cells. Our data highlight the importance of Vif in HIV-1 pathogenesis and also provide a unique tool to investigate the interaction of Vif and Apobec3G.     

Deletion of the GPG motif in the HIV type 1 V3 loop does not abrogate infection in all cells

The three amino acids glycine, proline, and glycine (GPG) constitute a conserved motif at the center of the V3 loop of HIV-1 surface glycoprotein 120. It has been indicated that deletion of this GPG motif is lethal for viral infectivity and abrogates the ability of the virus to form syncytia. In the present work, we studied the effects of GPG deletion on viral infectivity, cell tropism, syncytium formation, and initiation of apoptosis by constructing a mutant provirus based on the infectious clone pBRu-2. Successful infection and replication of GPG-deleted virus were detected in MT-2 cells, although the mutant virus showed lower infectivity. Infection could also be observed in the C8166, C91-PL, Molt-3, and THP-1 cell lines, and in PBMC-derived dendritic cells (DCs), but not in CEM-SS, HUT78, H9, Jurkat, and U937 cell lines or in PBMCs. Mutant virus also induced syncytia and apoptosis in the MT-2 cells. An intact GPG motif is probably necessary for unimpaired induction of fusion in some HIV-1-permissive cells. However, once the virus enters the cells, the GPG sequence does not seem to be indispensable for syncytium formation or apoptosis induction in MT-2 cells. Our data also imply that cell surface molecules other than CD4 and CXCR4 may be involved in entry of the GPG-deleted virus.     

Human immunodeficiency virus 1 reservoir in CD4+ T cells is restricted to certain V beta subsets.

The human immunodeficiency virus 1 (HIV-1) replicates more efficiently in T-cell lines expressing T-cell receptors derived from certain V beta genes, V beta 12 in particular, suggesting the effects of a superantigen. The targeted V beta 12 subset was not deleted in HIV-1-infected patients. It was therefore possible that it might represent an in vivo viral reservoir. Viral load was assessed by quantitative PCR with gag primers and with an infectivity assay to measure competent virus. It was shown that the tiny V beta 12 subset (1-2% of T cells) often has a higher viral load than other V beta subsets in infected patients. Selective HIV-1 replication in V beta 12 cells was also observed 6-8 days after in vitro infection of peripheral blood lymphocytes from normal, HIV-1 negative donors. Viral replication in targeted V beta subsets may serve to promote a biologically relevant viral reservoir.