HIV-1 Infection of T Cells and Macrophages Are Differentially Modulated by Virion-Associated Hck: A Nef-Dependent Phenomenon

The proline repeat motif (PxxP) of Nef is required for interaction with the SH3 domains of macrophage-specific Src kinase Hck. However, the implication of this interaction for viral replication and infectivity in macrophages and T lymphocytes remains unclear. Experiments in HIV-1 infected macrophages confirmed the presence of a Nef:Hck complex which was dependent on the Nef proline repeat motif. The proline repeat motif of Nef also enhanced both HIV-1 infection and replication in macrophages, and was required for incorporation of Hck into viral particles. Unexpectedly, wild-type Hck inhibited infection of macrophages, but Hck was shown to enhance infection of primary T lymphocytes. These results indicate that the interaction between Nef and Hck is important for Nef-dependent modulation of viral infectivity. Hck-dependent enhancement of HIV-1 infection of T cells suggests that Nef-Hck interaction may contribute to the spread of HIV-1 infection from macrophages to T cells by modulating events in the producer cell, virion and target cell.     

Guanidine alkaloid analogs as inhibitors of HIV-1 Nef interactions with p53, actin, and p56lck

With current anti-HIV treatments targeting only 4 of the 15 HIV proteins, many potential viral vulnerabilities remain unexploited. We report small-molecule inhibitors of the HIV-1 protein Nef. In addition to expanding the anti-HIV arsenal, small-molecule inhibitors against untargeted HIV proteins could be used to dissect key events in the HIV lifecycle. Numerous incompletely characterized interactions between Nef and cellular ligands, for example, present a challenge to understanding molecular events during HIV progression to AIDS. Assays with phage-displayed Nef from HIV(NL4-3) were used to identify a series of guanidine alkaloid-based inhibitors of Nef interactions with p53, actin, and p56(lck). The guanidines, synthetic analogs of batzellidine and crambescidin natural products, inhibit the Nef-ligand interactions with IC(50) values in the low micromolar range. In addition, sensitive in vivo assays for Nef inhibition are reported. Although compounds that are effective in vitro proved to be too cytotoxic for cellular assays, the reported Nef inhibitors provide proof-of-concept for disrupting a new HIV target and offer useful leads for drug development.     

The Nef-infectivity enigma: mechanisms of enhanced lentiviral infection

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.     

Influence of Different Glycoproteins and of the Virion Core on SERINC5 Antiviral Activity

Host plasma membrane protein SERINC5 is incorporated into budding retrovirus particles where it blocks subsequent entry into susceptible target cells. Three structurally unrelated proteins encoded by diverse retroviruses, human immunodeficiency virus type 1 (HIV-1) Nef, equine infectious anemia virus (EIAV) S2, and ecotropic murine leukemia virus (MLV) GlycoGag, disrupt SERINC5 antiviral activity by redirecting SERINC5 from the site of virion assembly on the plasma membrane to an internal RAB7+ endosomal compartment. Pseudotyping retroviruses with particular glycoproteins, e.g., vesicular stomatitis virus glycoprotein (VSV G), renders the infectivity of particles resistant to inhibition by virion-associated SERINC5. To better understand viral determinants for SERINC5-sensitivity, the effect of SERINC5 was assessed using HIV-1, MLV, and Mason-Pfizer monkey virus (M-PMV) virion cores, pseudotyped with glycoproteins from Arenavirus, Coronavirus, Filovirus, Rhabdovirus, Paramyxovirus, and Orthomyxovirus genera. SERINC5 restricted virions pseudotyped with glycoproteins from several retroviruses, an orthomyxovirus, a rhabdovirus, a paramyxovirus, and an arenavirus. Infectivity of particles pseudotyped with HIV-1, amphotropic-MLV (A-MLV), or influenza A virus (IAV) glycoproteins, was decreased by SERINC5, whether the core was provided by HIV-1, MLV, or M-PMV. In contrast, particles pseudotyped with glycoproteins from M-PMV, parainfluenza virus 5 (PIV5), or rabies virus (RABV) were sensitive to SERINC5, but only with particular retroviral cores. Resistance to SERINC5 did not correlate with reduced SERINC5 incorporation into particles, route of viral entry, or absolute infectivity of the pseudotyped virions. These findings indicate that some non-retroviruses may be sensitive to SERINC5 and that, in addition to the viral glycoprotein, the retroviral core influences sensitivity to SERINC5.     

HIV Nef: role in pathogenesis and viral fitness

Conserved in all primate lentivirus genomes, Nef promotes viral replication and infectivity, influences the trafficking of a large number of surface receptors and interferes with TCR signalling, consequently modulating T-cell activation. In vivo observations with Long Term Non-Progressors harbouring a Nef-defective HIV and vaccination studies with Nef-deleted SIV in Rhesus macaques have shown a prominent role for Nef in lentiviral pathogenesis. Here we review the functions of Nef involved in viral replication and infectivity and speculate on a possible role for Nef in HIV fitness.     

Recent insights into HIV accessory proteins

HIV produces structural, regulatory, and accessory proteins during viral replication in host cells. The accessory proteins include Nef, viral infectivity factor (Vif), viral protein R, and viral protein U or viral protein X. Although these accessory proteins are generally dispensable for viral replication in vitro, they are essential for viral pathogenesis in vivo. Consequently, there has been much interest in understanding how these accessory proteins function because this research may yield new antiviral targets to curb HIV pathogenesis in vivo. Therefore, this review highlights recent advances in understanding the HIV accessory proteins and emphasizes breakthrough insights into the elusive Vif protein and potential new targets for therapeutic intervention.     

A New Generation of Functional Tagged Proteins for HIV Fluorescence Imaging

During the last decade, there was a marked increase in the development of tools and techniques to study the molecular mechanisms of the HIV replication cycle by using fluorescence microscopy. Researchers often apply the fusion of tags and fluorophores to viral proteins, surrogate proteins, or dyes to follow individual virus particles while they progress throughout infection. The inclusion of such fusion motifs or surrogates frequently disrupts viral infectivity or results in a change of the wild-type phenotype. Here, we detail the construction and functional characterization of two new constructs where we fused fluorescent proteins to the N-terminus of HIV-1 Integrase. In the first, IN is recruited into assembling particles via a codon optimized Gag to complement other viral constructs, while the second is fused to a Gag-Pol expression vector fully capable of integration. Our data shows that N-terminal tagged IN is functional for integration by both recovery of integration of catalytically inactive IN and by the successful infectivity of viruses carrying only labeled IN. These tools will be important to study the individual behavior of viral particles and associate such behavior to infectivity.     

Superresolution imaging of HIV in infected cells with FlAsH-PALM

Imaging protein assemblies at molecular resolution without affecting biological function is a long-standing goal. The diffraction-limited resolution of conventional light microscopy (∼200–300 nm) has been overcome by recent superresolution (SR) methods including techniques based on accurate localization of molecules exhibiting stochastic fluorescence; however, SR methods still suffer important restrictions inherent to the protein labeling strategies. Antibody labels are encumbered by variable specificity, limited commercial availability and affinity, and are mostly restricted to fixed cells. Fluorescent protein fusions, though compatible with live cell imaging, substantially increase protein size and can interfere with their biological activity. We demonstrate SR imaging of proteins tagged with small tetracysteine motifs and the fluorescein arsenical helix binder (FlAsH-PALM). We applied FlAsH-PALM to image the integrase enzyme (IN) of HIV in fixed and living cells under experimental conditions that fully preserved HIV infectivity. The obtained resolution (∼30 nm) allowed us to characterize the distribution of IN within virions and intracellular complexes and to distinguish different HIV structural populations based on their morphology. We could thus discriminate ∼100 nm long mature conical cores from immature Gag shells and observe that in infected cells cytoplasmic (but not nuclear) IN complexes display a morphology similar to the conical capsid. Together with the presence of capsid proteins, our data suggest that cytoplasmic IN is largely present in intact capsids and that these can be found deep within the cytoplasm. FlAsH-PALM opens the door to in vivo SR studies of microbial complexes within host cells and may help achieve truly molecular resolution.     

Role of Transportin-SR2 in HIV-1 Nuclear Import

The HIV replication cycle depends on the interaction of viral proteins with proteins of the host. Unraveling host–pathogen interactions during the infection is of great importance for understanding the pathogenesis and the development of antiviral therapies. To date HIV uncoating and nuclear import are the most debated steps of the HIV-1 replication cycle. Despite numerous studies during past decades, there is still much controversy with respect to the identity and the role of viral and host factors involved in these processes. In this review, we provide a comprehensive overview on the role of transportin-SR2 as a host cell factor during active nuclear transport.     

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.     

HIV-1 Integrase-DNA Recognition Mechanisms

Integration of a reverse transcribed DNA copy of the HIV viral genome into the host chromosome is essential for virus replication. This process is catalyzed by the virally encoded protein integrase. The catalytic activities, which involve DNA cutting and joining steps, have been recapitulated in vitro using recombinant integrase and synthetic DNA substrates. Biochemical and biophysical studies of these model reactions have been pivotal in advancing our understanding of mechanistic details for how IN interacts with viral and target DNAs, and are the focus of the present review.     

HIV-1 Nef increases T cell activation in a stimulus-dependent manner.

Lentiviral Nef increases viral replication in vivo, plays a direct role in pathogenesis, and increases viral particle infectivity. We now find that HIV Nef also increases the activation of T cells, a cellular state required for optimal viral replication. This enhancement is stimulant-dependent. As defined by IL-2 generation, activation of T cells stimulated with classical mitogens [phorbol 12-myristate 13-acetate (PMA) + anti-CD3, PMA + phytohemagglutinin, and PMA + ionomycin] is unaffected by the expression of Nef. However, Nef increases IL-2 secretion when cells are stimulated through the T cell receptor and the costimulus receptor (CD28). This increase in activation, which depends on Nef myristylation, is caused by an increase in the number of cells reaching full activation and not by an increase in the amount of IL-2 secreted per cell. These findings demonstrate that Nef lowers the threshold of the dual-receptor T cell activation pathway. The capacity of Nef to increase T cell activity may be very important in vivo when Nef is the predominant or the only viral gene product expressed.     

Nef increases the synthesis of and transports cholesterol to lipid rafts and HIV-1 progeny virions

HIV buds from lipid rafts and requires cholesterol for its egress from and entry into cells. Viral accessory protein Nef plays a major role in this process. In this study, it not only increased the biosynthesis of lipid rafts and viral particles with newly synthesized cholesterol, but also enriched them. Furthermore, via the consensus cholesterol recognition motif at its C terminus, Nef bound cholesterol. When this sequence was mutated, Nef became unable to transport newly synthesized cholesterol into lipid rafts and viral particles. Interestingly, although its levels in lipid rafts were not affected, this mutant Nef protein was poorly incorporated into viral particles, and viral infectivity decreased dramatically. Thus, Nef also transports newly synthesized cholesterol to the site of viral budding. As such, it provides essential building blocks for the formation of viruses that replicate optimally in the host.     

Spotlight on HIV-1 Nef: SERINC3 and SERINC5 Identified as Restriction Factors Antagonized by the Pathogenesis Factor

The Nef protein is an accessory gene product encoded by human immunodeficiency virus types 1 and 2 (HIV-1/-2) and simian immunodeficiency virus (SIV) that boosts virus replication in the infected host and accelerates disease progression. Unlike the HIV-1 accessory proteins Vif, Vpr and Vpu, Nef was, until recently, not known to antagonize the antiviral activity of a host cell restriction factor. Two recent reports now describe the host cell proteins serine incorporator 3 and 5 (SERINC3 and SERINC5) as potent inhibitors of HIV-1 particle infectivity and demonstrate that Nef counteracts these effects. These findings establish SERINC3/5 as restrictions to HIV replication in human cells and define a novel activity for the HIV pathogenesis factor Nef.     

HIV Type 1 Nef is released from infected cells in CD45(+) microvesicles and is present in the plasma of HIV-infected individuals

HIV-1 Nef has been demonstrated to be integral for viral persistence, infectivity, and the acceleration of disease pathogenesis (AIDS) in humans. Nef has also been detected in the plasma of HIV-infected individuals and is released from infected cells. The form in which Nef is released from infected cells is unknown. However, Nef is a myristoylated protein and has been shown to interact with the intracellular vesicular trafficking network. Here we show that Nef is released in CD45-containing microvesicles. This microvesicular Nef (mvNef) is detected in the plasma of HIV-infected individuals at relatively high concentrations (10?ng/ml). It is also present in tissue culture supernatants of Jurkat cells infected with HIV(MN). Interestingly, plasma mvNef levels in HIV(+) patients did not significantly correlate with viral load or CD4 count. Microvesicular Nef levels persisted in the plasma of HIV-infected individuals despite the use of antiretroviral therapy, even in individuals with undetectable viral loads. Using cell lines, we found Nef microvesicles induce apoptosis in Jurkat T-lymphocytes but had no observed effect on the U937 monocytic cell line. Given the large amount of mvNef present in the plasma of HIV-infected individuals, the apoptotic effect of mvNef on T cells, and the observed functions of extracellular soluble Nef in vitro, it seems likely that in vivo mvNef may play a significant role in the pathogenesis of AIDS.