Enhancement Of Virus Infection Research Articles

Antibody-Dependent Enhancement of Marburg Virus Infection

Marburg virus (MARV) and Ebola virus (EBOV) cause severe hemorrhagic fever in primates. Earlier studies demonstrated that antibodies to particular epitopes on the glycoprotein (GP) of EBOV enhanced virus infectivity in vitro. To investigate this antibody-dependent enhancement (ADE) in MARV infection, we produced mouse antisera and monoclonal antibodies (mAbs) to the GPs of MARV strains Angola and Musoke. The infectivity of vesicular stomatitis virus pseudotyped with Angola GP in K562 cells was significantly enhanced in the presence of Angola GP antisera, whereas only minimal ADE activity was seen with Musoke GP antisera. This difference correlated with the percentage of hybridoma clones producing infectivity-enhancing mAbs. Using mAbs to MARV GP, we identified 3 distinct ADE epitopes in the mucinlike region on Angola GP. Interestingly, some of these antibodies bound to both Angola and Musoke GPs but showed significantly higher ADE activity for strain Angola. ADE activity depended on epitopes in the mucinlike region and glycine at amino acid position 547, present in the Angola but absent in the Musoke GP. These results suggest a possible link between ADE and MARV pathogenicity and provide new insights into the mechanisms underlying ADE entry of filoviruses.

Improved lentiviral gene transfer into human embryonic stem cells grown in co-culture with murine feeder and stroma cells

Genetic modification of human embryonic stem cells (hESCs) using biophysical DNA transfection methods are hampered by the very low single cell survival rate and cloning efficiency of hESCs. Lentiviral gene transfer strategies are widely used to genetically modify hESCs but limited transduction efficiencies in the presence of feeder or stroma cells present problems, particularly if vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped viral particles are applied. Here, we investigated whether the recently described semen derived enhancer of virus infection (SEVI) and alternative viral envelope proteins derived from either Gibbon ape leukaemia virus (GALV) or feline leukaemia virus (RD114) are applicable for transducing hESCs during co-culture with feeder or stroma cells. Our first set of experiments demonstrates that SEVI has no toxic effect on murine or hESCs and that exposure to SEVI does not interfere with the pluripotency-associated phenotype. Focusing on hESCs, we were able to further demonstrate that SEVI increases the transduction efficiencies of GALV and RD114 pseudotyped lentiviral vectors. More importantly, aiming at targeted differentiation of hESCs into functional somatic cell types, GALV pseudotyped lentiviral particles could efficiently and exclusively transduce hESCs grown in co-culture with OP9-GFP stroma cells (which were often used to induce differentiation into haematopoietic derivatives).

Ligation of Fc gamma receptor IIB inhibits antibody-dependent enhancement of dengue virus infection

The interaction of antibodies, dengue virus (DENV), and monocytes can result in either immunity or enhanced virus infection. These opposing outcomes of dengue antibodies have hampered dengue vaccine development. Recent studies have shown that antibodies neutralize DENV by either preventing virus attachment to cellular receptors or inhibiting viral fusion intracellularly. However, whether the antibody blocks attachment or fusion, the resulting immune complexes are expected to be phagocytosed by Fc gamma receptor (FcγR)-bearing cells and cleared from circulation. This suggests that only antibodies that are able to block fusion intracellularly would be able to neutralize DENV upon FcγR-mediated uptake by monocytes whereas other antibodies would have resulted in enhancement of DENV replication. Using convalescent sera from dengue patients, we observed that neutralization of the homologous serotypes occurred despite FcγR-mediated uptake. However, FcγR-mediated uptake appeared to be inhibited when neutralized heterologous DENV serotypes were used instead. We demonstrate that this inhibition occurred through the formation of viral aggregates by antibodies in a concentration-dependent manner. Aggregation of viruses enabled antibodies to cross-link the inhibitory FcγRIIB, which is expressed at low levels but which inhibits FcγR-mediated phagocytosis and hence prevents antibody-dependent enhancement of DENV infection in monocytes.

Mechanisms of HIV-1 Nef Function and Intracellular Signaling

Advances in the last several years have enhanced mechanistic understanding of Nef-induced CD4 and MHCI downregulation and have suggested a new paradigm for analyzing Nef function. In both of these cases, Nef acts by forming ternary complexes with significant contributions to stability imparted by non-canonical interactions. The mutational analyses and binding assays that have led to these conclusions are discussed. The recent progress has been dependent on conservative mutations and multi-protein binding assays. The poorly understood Nef functions of p21 activated protein kinase (PAK2) activation, enhancement of virion infectivity, and inhibition of immunoglobulin class switching are also likely to involve ternary complexes and non-canonical interactions. Hence, investigation of these latter Nef functions should benefit from a similar approach. Six historically used alanine substitutions for determining structure-function relationships of Nef are discussed. These are M20A, E62A/E63A/E64A/E65A (AAAA), P72A/P75A (AXXA), R106A, L164A/L165A, and D174A/D175A. Investigations of less-disruptive mutations in place of AAAA and AXXA have led to different interpretations of mechanism. Two recent examples of this alternate approach, F191I for studying PAK2 activation and D123E for the critical residue D123 are discussed. The implications of the new findings and the resulting new paradigm for Nef structure-function are discussed with respect to creating a map of Nef functions on the protein surface. We report the results of a PPI-Pred analysis for protein-protein interfaces. There are three predicted patches produced by the analysis which describe regions consistent with the currently known mutational analyses of Nef function.

Blocking Semen-Mediated Enhancement of HIV Infection by Amyloid-Binding Small Molecules

Evaluation of: Olsen JS, Brown C, Capule CC et al.: Amyloid-binding small molecules efficiently block SEVI (semen-derived enhancer of virus infection)- and semen-mediated enhancement of HIV-1 infection. J. Biol. Chem. 285(46), 35488–35496 (2010). Sexual intercourse is the major route for the spread of the AIDS pandemic and semen represents the main vector for HIV-1 transmission. It is under debate whether semen enhances or inhibits HIV-1 infection. However, accumulating evidence suggests that semen contains amyloid aggregates (termed ‘semen-derived enhancer of virus infection’ [SEVI]) that boost infection and thus represent a promising target for the prevention of HIV-1 transmission. Here, Olsen and colleagues demonstrate that BTA-EG6, a derivative of the amyloid-specific dye thioflavin T, binds SEVI fibrils. This interaction blocks the ability of SEVI to enhance virus infection by preventing the association of SEVI with target cells. Importantly, the authors also demonstrate that BTA-EG6 inhibits semen-mediated enhancement of HIV-1 infection without causing inflammation or toxicity in cervical cells. These results suggest that small amyloid-binding molecules that block semen-mediated enhancement of HIV-1 infection are a useful supplement to microbicides for preventing sexual transmission of the virus.

Assembly and Replication of HIV-1 in T Cells with Low Levels of Phosphatidylinositol-(4,5)-Bisphosphate

HIV-1 Gag assembles into virus particles predominantly at the plasma membrane (PM). Previously, we observed that phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] is essential for Gag binding to the plasma membrane and virus release in HeLa cells. In the current study, we found that PI(4,5)P(2) also facilitates Gag binding to the PM and efficient virus release in T cells. Notably, serial passage of HIV-1 in an A3.01 clone that expresses polyphosphoinositide 5-phosphatase IV (5ptaseIV), which depletes cellular PI(4,5)P(2), yielded an adapted mutant with a Leu-to-Arg change at matrix residue 74 (74LR). Virus replication in T cells expressing 5ptaseIV was accelerated by the 74LR mutation relative to replication of wild type HIV-1 (WT). This accelerated replication of the 74LR mutant was not due to improved virus release. In control T cells, the 74LR mutant releases virus less efficiently than does the WT, whereas in cells expressing 5ptaseIV, the WT and the 74LR mutant are similarly inefficient in virus release. Unexpectedly, we found that the 74LR mutation increased virus infectivity and compensated for the inefficient virus release. Altogether, these results indicate that PI(4,5)P(2) is essential for Gag-membrane binding, targeting of Gag to the PM, and efficient virus release in T cells, which in turn likely promotes efficient virus spread in T cell cultures. In T cells with low PI(4,5)P(2) levels, however, the reduced virus particle production can be compensated for by a mutation that enhances virus infectivity.

Herpesviruses Enhance Fibrinogen Clot Lysis.

AbstractAbstract 1153 Background:Members of the Herpesvirus family have been implicated in vascular disease. To explain the correlation on a molecular basis we have shown that the virus envelope contains anionic phospholipid derived from host cells, and proteins encoded by the host (tissue factor) as well as the viral (glycoprotein C) genomes, which initiate blood coagulation. This suggests that virus infection should be a strong independent predictor of vascular disease. Nevertheless, the clinical correlation is relatively weak, becoming more significant in combination with other risk factors. To explain this discrepancy, the current work is based on our additional report that at least one Herpesvirus (cytomegalovirus (CMV)) has host-genome-encoded annexin II on its surface. Annexin II is known to accelerate tissue plasminogen activator (tPA)-mediated activation of plasminogen to plasmin because of C-terminal lysines that interact with both plasminogen and tPA. Plasmin is the primary fibrinolytic protease, and is necessary for physiological clot dissolution. Thus, these viruses may facilitate clearance of the fibrin they generate. Hypothesis:We hypothesize that Herpesviruses enhance tPA-mediated plasmin generation and this mechanism correlates to the presence of annexin II on the virus. Methods:Purified herpes simplex virus type 1 (HSV1) and 2 (HSV2) and CMV were quantified by electron microscopy. Annexin II expression varies between cell types, therefore HSV1 was propagated in several cell lines. Virus-dependent plasmin generation was followed in the presence of purified plasminogen and tPA using a chromogenic assay. The contribution of viruses to fibrin clot lysis using purified proteins was investigated by light scattering. Plasminogen-conjugated horse radish peroxidase (plasminogen-HRP) and western blots were used to identify plasminogen-binding species and annexin II associated with the virus, respectively. The effect of plasmin-mediated signalling on virus infection was determined using cytopathic plaque assays. Results:Chromogenic experiments demonstrated that HSV1, HSV2 and CMV enhanced plasminogen activation in a dose-dependent manner by up to 5-fold, regardless of the parental cell line. Prolonged incubation confirmed the requirement for exogenous tPA. Plasminogen-HRP bound to a number of virus-associated proteins and was shown to be C-terminal lysine-dependent by complete inhibition with epsilon-aminocaproic acid (EACA). Annexin II was demonstrated to be associated with purified HSV1 cultured in different cells except when propagated in a melanoma (A7) line that did not express annexin II. An annexin II antibody inhibited binding of plasminogen-HRP to viral annexin II. HSV1, HSV2 and CMV accelerated fibrin clot lysis, which was inhibited in the presence of EACA and aprotinin, a plasmin inhibitor. However, in contrast to the chromogenic experiment for plasmin generation, each virus also exhibited a clot lysis mechanism independent of added tPA. As we have previously identified for thrombin, incubation of host cells with purified plasmin during inoculation enhanced virus infection by over 3-fold. Conclusion:Cumulatively these data demonstrate that HSV1, HSV2 and CMV accelerate tPA- mediated plasmin generation in the absence of fibrin and identify annexin II as one of several plasminogen binding partners. These viruses furthermore enhance the rate of fibrin clot lysis. However, in the presence of fibrin, purified HSV1, HSV2 and CMV are capable of facilitating clot dissolution in the absence of exogenous tPA. The molecular basis for this novel mechanism is not yet known, but requires plasminogen activation. The finding that purified plasmin enhances infection suggests these viruses may have evolved to initiate plasmin generation. Hence, the virus-mediated activation of fibrinolysis may compensate for its ability to trigger coagulation and attenuate potential contributions as an independent predictor of vascular disease. Disclosures:No relevant conflicts of interest to declare.

Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX

Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.

Insulin degrading enzyme induces a conformational change in varicella-zoster virus gE, and enhances virus infectivity and stability.

Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for virus infectivity and binds to a cellular receptor, insulin-degrading enzyme (IDE), through its unique amino terminal extracellular domain. Previous work has shown IDE plays an important role in VZV infection and virus cell-to-cell spread, which is the sole route for VZV spread in vitro. Here we report that a recombinant soluble IDE (rIDE) enhances VZV infectivity at an early step of infection associated with an increase in virus internalization, and increases cell-to-cell spread. VZV mutants lacking the IDE binding domain of gE were impaired for syncytia formation and membrane fusion. Pre-treatment of cell-free VZV with rIDE markedly enhanced the stability of the virus over a range of conditions. rIDE interacted with gE to elicit a conformational change in gE and rendered it more susceptible to proteolysis. Co-incubation of rIDE with gE modified the size of gE. We propose that the conformational change in gE elicited by IDE enhances infectivity and stability of the virus and leads to increased fusogenicity during VZV infection. The ability of rIDE to enhance infectivity of cell-free VZV over a wide range of incubation times and temperatures suggests that rIDE may be useful for increasing the stability of varicella or zoster vaccines.

A Low-Molecular-Weight Entry Inhibitor of both CCR5- and CXCR4-Tropic Strains of Human Immunodeficiency Virus Type 1 Targets a Novel Site on gp41

A low-molecular-weight human immunodeficiency virus type 1 (HIV-1) inhibitor, PF-68742 (molecular weight, 573), has been identified in a high-throughput screen for compounds that block HIV-1 envelope glycoprotein (Env)-mediated fusion. The compound is shown to be potent against R5 and X4 isolates in both cell-cell fusion and antiviral assays (50% effective concentrations of approximately 0.1 to 1 muM). Postfusion and HIV-1 pseudotyping control experiments confirm that PF-68742 is an entry inhibitor with Env as the specific target for antiviral action. PF-68742 was not able to block binding of monomeric gp120 to soluble CD4 or the binding of gp120:CD4 complexes to cell-associated CCR5, thus distinguishing PF-68742 from described gp120 antagonists and coreceptor binders. Escape variants of HIV-1(NL4-3) were selected, and all resistant viruses were found to contain a common G514R (HxB2 numbering) mutation in Env, located proximal to the furin cleavage site in the fusion peptide of gp41. When introduced into wild-type NL4-3 gp41, G514R conferred resistance to PF-68742. Resistance via G514R is shown to be associated with enhancement of virion infectivity by PF-68742 that may result from altered properties of inhibitor-bound Env, rather than from a loss of compound binding. Wild-type viruses and those with substitutions in the disulfide loop (DSL) region of gp41 were also examined for PF-68742 sensitivity. Here, complete resistance to PF-68742 was found to occur through changes outside of position 514, including in the gp41 DSL region. The results highlight PF-68742 as a starting point for novel therapies against HIV-1 and provide new insights into models of Env-mediated fusion.

Regulation of Microtubule Dynamics through Phosphorylation on Stathmin by Epstein-Barr Virus Kinase BGLF4

Stathmin is an important microtubule (MT)-destabilizing protein, and its activity is differently attenuated by phosphorylation at one or more of its four phosphorylatable serine residues (Ser-16, Ser-25, Ser-38, and Ser-63). This phosphorylation of stathmin plays important roles in mitotic spindle formation. We observed increasing levels of phosphorylated stathmin in Epstein-Barr virus (EBV)-harboring lymphoblastoid cell lines (LCLs) and nasopharyngeal carcinoma (NPC) cell lines during the EBV lytic cycle. These suggest that EBV lytic products may be involved in the regulation of stathmin phosphorylation. BGLF4 is an EBV-encoded kinase and has similar kinase activity to cdc2, an important kinase that phosphorylates serine residues 25 and 38 of stathmin during mitosis. Using an siRNA approach, we demonstrated that BGLF4 contributes to the phosphorylation of stathmin in EBV-harboring NPC. Moreover, we confirmed that BGLF4 interacts with and phosphorylates stathmin using an in vitro kinase assay and an in vivo two-dimensional electrophoresis assay. Interestingly, unlike cdc2, BGLF4 was shown to phosphorylate non-proline directed serine residues of stathmin (Ser-16) and it mediated phosphorylation of stathmin predominantly at serines 16, 25, and 38, indicating that BGLF4 can down-regulate the activity of stathmin. Finally, we demonstrated that the pattern of MT organization was changed in BGLF4-expressing cells, possibly through phosphorylation of stathmin. In conclusion, we have shown that a viral Ser/Thr kinase can directly modulate the activity of stathmin and this contributes to alteration of cellular MT dynamics and then may modulate the associated cellular processes.

Quantitative Comparison of HTLV-1 and HIV-1 Cell-to-Cell Infection with New Replication Dependent Vectors

We have developed an efficient method to quantify cell-to-cell infection with single-cycle, replication dependent reporter vectors. This system was used to examine the mechanisms of infection with HTLV-1 and HIV-1 vectors in lymphocyte cell lines. Effector cells transfected with reporter vector, packaging vector, and Env expression plasmid produced virus-like particles that transduced reporter gene activity into cocultured target cells with zero background. Reporter gene expression was detected exclusively in target cells and required an Env-expression plasmid and a viral packaging vector, which provided essential structural and enzymatic proteins for virus replication. Cell-cell fusion did not contribute to infection, as reporter protein was rarely detected in syncytia. Coculture of transfected Jurkat T cells and target Raji/CD4 B cells enhanced HIV-1 infection two fold and HTLV-1 infection ten thousand fold in comparison with cell-free infection of Raji/CD4 cells. Agents that interfere with actin and tubulin polymerization strongly inhibited HTLV-1 and modestly decreased HIV-1 cell-to-cell infection, an indication that cytoskeletal remodeling was more important for HTLV-1 transmission. Time course studies showed that HTLV-1 transmission occurred very rapidly after cell mixing, whereas slower kinetics of HIV-1 coculture infection implies a different mechanism of infectious transmission. HTLV-1 Tax was demonstrated to play an important role in altering cell-cell interactions that enhance virus infection and replication. Interestingly, superantigen-induced synapses between Jurkat cells and Raji/CD4 cells did not enhance infection for either HTLV-1 or HIV-1. In general, the dependence on cell-to-cell infection was determined by the virus, the effector and target cell types, and by the nature of the cell-cell interaction.

Enhancement of the Influenza A Hemagglutinin (HA)-Mediated Cell-Cell Fusion and Virus Entry by the Viral Neuraminidase (NA)

BackgroundThe major role of the neuraminidase (NA) protein of influenza A virus is related to its sialidase activity, which disrupts the interaction between the envelope hemagglutin (HA) protein and the sialic acid receptors expressed at the surface of infected cells. This enzymatic activity is known to promote the release and spread of progeny viral particles following their production by infected cells, but a potential role of NA in earlier steps of the viral life cycle has never been clearly demonstrated. In this study we have examined the impact of NA expression on influenza HA-mediated viral membrane fusion and virion infectivity.Methodology/Principal FindingsThe role of NA in the early stages of influenza virus replication was examined using a cell-cell fusion assay that mimics HA-mediated membrane fusion, and a virion infectivity assay using HIV-based pseudoparticles expressing influenza HA and/or NA proteins. In the cell-cell fusion assay, which bypasses the endocytocytosis step that is characteristic of influenza virus entry, we found that in proper HA maturation conditions, NA clearly enhanced fusion in a dose-dependent manner. Similarly, expression of NA at the surface of pseudoparticles significantly enhanced virion infectivity. Further experiments using exogeneous soluble NA revealed that the most likely mechanism for enhancement of fusion and infectivity by NA was related to desialylation of virion-expressed HA.Conclusion/SignificanceThe NA protein of influenza A virus is not only required for virion release and spread but also plays a critical role in virion infectivity and HA-mediated membrane fusion.

Modulation of PML protein expression regulates JCV infection

JC virus (JCV) is a human polyomavirus that infects the majority of the human population worldwide. It is responsible for the fatal demyelinating disease Progressive Multifocal Leukoencephalopathy. JCV binds to cells using the serotonin receptor 5-HT(2A)R and alpha(2-6)- or alpha(2-3)-linked sialic acid. It enters cells using clathrin-dependent endocytosis and traffics to the early endosome and possibly to the endoplasmic reticulum. Viral DNA is then delivered to the nucleus where transcription, replication, and assembly of progeny occur. We found that the early regulatory protein large T antigen accumulates in microdomains in the nucleus adjacent to ND-10 or PML domains. This observation prompted us to explore the role of these domains in JCV infection. We found that a reduction of nuclear PML enhanced virus infection and that an increase in nuclear PML reduced infection. Infection with JCV did not directly modulate nuclear levels of PML but our data indicate that a host response involving interferon beta is likely to restrict virus infection by increasing nuclear PML.

Conservation of Nef function across highly diverse lineages of SIVsmm

BackgroundSIVsmm is a simian immunodeficiency virus that persists efficiently without causing disease in naturally infected sooty mangabeys (SMs) but induces AIDS upon cross-species transmission to humans and macaques. Current phylogenetic data indicate that SIVsmm strains comprise a highly diverse group of viruses that can be subdivided into different lineages. Since only certain SIVsmm strains have successfully crossed the species barrier to humans and macaques, the question has been raised whether there are lineage specific differences in SIVsmm biology. In the present study we examined whether representatives of five different SIVsmm lineages show differences in the function of the accessory Nef protein, which plays an important role in viral persistence, transmission and pathogenesis.ResultsWe found that nef alleles from all SIVsmm lineages down-modulated CD4, MHC-I, CD28 and CD3 and up-regulated the invariant chain (Ii) associated with immature MHC-II molecules in human-derived cells. Moreover, they generally suppressed the responsiveness of virally infected T cells to activation, enhanced virion infectivity and promoted virus replication in human peripheral blood mononuclear cells. The functional activity of these nef alleles in the various assays varied substantially between different strains of SIVsmm but quantitative analyses did not reveal any significant lineage-specific differences in Nef function.ConclusionNef alleles from different lineages of SIVsmm do not require adaptive changes to be functionally active in human cells. Strain rather than lineage-specific differences in Nef function may impact the virological and immunological feature of SIVsmm in SMs and possibly affected viral fitness and pathogenicity in human and macaque hosts.

Co‐infection of respiratory bacterium with severe acute respiratory syndrome coronavirus induces an exacerbated pneumonia in mice

ABSTRACTSARS‐CoV grows in a variety of tissues that express its receptor, although the mechanism for high replication in the lungs and severe respiratory illness is not well understood. We recently showed that elastase enhances SARS‐CoV infection in cultured cells, which suggests that SARS development may be due to elastase‐mediated, enhanced SARS‐CoV infection in the lungs. To explore this possibility, we examined whether co‐infection of mice with SARS‐CoV and Pp, a low‐pathogenic bacterium which elicits elastase production in the lungs, induces exacerbation of pneumonia. Mice co‐infected with SARS‐CoV and Pp developed severe respiratory disease with extensive weight loss, resulting in a 33∼90% mortality rate. Mice with exacerbated pneumonia showed enhanced virus infection in the lungs and histopathological lesions similar to those found in human SARS cases. Intranasal administration of LPS, another elastase inducer, showed an effect similar to that of Pp infection. Thus, this study shows that exacerbated pneumonia in mice results from co‐infection with SARS‐CoV and a respiratory bacterium that induces elastase production in the lungs, suggesting a possible role for elastase in the exacerbation of pneumonia.

Nef enhances HIV-1 infectivity via association with the virus assembly complex

The HIV-1 accessory protein Nef enhances virus infectivity by facilitating an early post-entry step of infection. Nef acts in the virus producer cell, leading to a beneficial modification to HIV-1 particles. Nef itself is incorporated into HIV-1 particles, where it is cleaved by the viral protease during virion maturation. To probe the role of virion-associated Nef in HIV-1 infection, we generated a fusion protein consisting of the host protein cyclophilin A (CypA) linked to the amino terminus of Nef. The resulting CypA-Nef protein enhanced the infectivity of Nef-defective HIV-1 particles and was specifically incorporated into the virions via association with Gag during particle assembly. Pharmacologic or genetic inhibition of CypA-Nef binding to Gag prevented incorporation of CypA-Nef into virions and inhibited infectivity enhancement. Our results indicate that infectivity enhancement by Nef requires its association with a component of the assembling HIV-1 particle.

HIV-1 Nef: at the crossroads

The development of anti-virals has blunted the AIDS epidemic in the Western world but globally the epidemic has not been curtailed. Standard vaccines have not worked, and attenuated vaccines are not being developed because of safety concerns. Interest in attenuated vaccines has centered on isolated cases of patients infected with HIV-1 containing a deleted nef gene. Nef is a multifunctional accessory protein that is necessary for full HIV-1 virulence. Unfortunately, some patients infected with the nef-deleted virus eventually lose their CD4+ T cells to levels indicating progression to AIDS.This renders the possibility of an attenuated HIV-1 based solely on a deleted nef remote. In this review we discuss the knowledge gained both from the study of these patients and from in vitro investigations of Nef function to assess the possibility of developing new anti-HIV-1 drugs based on Nef. Specifically, we consider CD4 downregulation, major histocompatibility complex I downregulation, Pak2 activation, and enhancement of virion infectivity. We also consider the recent proposal that simian immunodeficiency viruses are non-pathogenic in their hosts because they have Nefs that downregulate CD3, but HIV-1 is pathogenic because its Nef fails to downregulate CD3. The possibility of incorporating the CD3 downregulation function into HIV-1 Nef as a therapeutic option is also considered. Finally, we conclude that inhibiting the CD4 downregulation function is the most promising Nef-targeted approach for developing a new anti-viral as a contribution to combating AIDS.

Nef-Mediated Enhancement of Virion Infectivity and Stimulation of Viral Replication Are Fundamental Properties of Primate Lentiviruses

Nef is a multifunctional accessory protein of primate lentiviruses. Recently, it has been shown that the ability of Nef to downmodulate CD4, CD28, and class I major histocompatibility complex is highly conserved between most or all primate lentiviruses, whereas Nef-mediated downregulation of T-cell receptor-CD3 was lost in the lineage that gave rise to human immunodeficiency virus type 1 (HIV-1). Whether or not other Nef activities are preserved between different groups of primate lentiviruses remained to be determined. Here, we show that nef genes from a large variety of HIVs and simian immunodeficiency viruses (SIVs) enhance virion infectivity and stimulate viral replication in human cells and/or in ex vivo infected human lymphoid tissue (HLT). Notably, nef alleles from unpassaged SIVcpz and SIVsmm enhanced viral infectivity, replication, and cytopathicity in cell culture and in ex vivo infected HLT as efficiently as those from HIV-1 and HIV-2, their human counterparts. Furthermore, nef genes from several highly divergent SIVs that have not been found in humans were also highly active in human cells and/or tissues. Thus, most primate lentiviral Nefs enhance virion infectivity and stimulate viral replication. Moreover, our data show that SIVcpz and SIVsmm Nefs do not require adaptive changes to perform these functions in human cells or tissues and support the idea that nef alleles from other primate lentiviruses would also be capable of promoting efficient virus spread in humans.

Association of Nef with p21-Activated Kinase 2 Is Dispensable for Efficient Human Immunodeficiency Virus Type 1 Replication and Cytopathicity in Ex Vivo-Infected Human Lymphoid Tissue

Interaction of the human immunodeficiency virus type 1 (HIV-1) Nef protein with p21-activated kinase 2 (PAK2) has been proposed to play a role in T-cell activation, viral replication, apoptosis, and progression to AIDS. However, these hypotheses were based on results obtained using Nef mutants impaired in multiple functions. Recently, it was reported that Nef residue F191 is specifically involved in PAK2 binding. However, only a limited number of Nef activities were investigated in these studies. To further evaluate the role of F191 in Nef function and to elucidate the biological relevance of Nef-PAK2 interaction, we performed a comprehensive analysis of HIV-1 Nef mutants carrying F191H and F191R mutations. We found that the F191H mutation reduces and the F191R mutation disrupts the association of Nef with PAK2. Both mutants upregulated the major histocompatibility complex II (MHC-II)-associated invariant chain and downregulated CD4, MHC-I, and CD28, although with reduced efficiency for the latter. Furthermore, the F191H/R changes neither affected the levels of interleukin-2 receptor expression and apoptosis of HIV-1-infected primary T cells nor reduced Nef-mediated induction of NFAT. Unexpectedly, the F191H change markedly reduced and the F191R mutation disrupted the ability of Nef to enhance virion infectivity in P4-CCR5 indicator cells but not in TZM-bl cells or peripheral blood mononuclear cells. Most importantly, all HIV-1 Nef mutants replicated efficiently and caused CD4+ T-cell depletion in ex vivo-infected human lymphoid tissue. Altogether, our data show that the interaction of Nef with PAK2 does not play a major role in T-cell activation, viral replication, and apoptosis.