Human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus isolated from a macaque monkey (SIVmac) are assumed to have originated from simian immunodeficiency virus isolated from sooty mangabey (SIVsm). Despite their close similarity in genome structure, HIV-2 and SIVmac show different sensitivities to TRIM5α, a host restriction factor against retroviruses. The replication of HIV-2 strains is potently restricted by rhesus (Rh) monkey TRIM5α, while that of SIVmac strain 239 (SIVmac239) is not. Viral capsid protein is the determinant of this differential sensitivity to TRIM5α, as the HIV-2 mutant carrying SIVmac239 capsid protein evaded Rh TRIM5α-mediated restriction. However, the molecular determinants of this restriction mechanism are unknown. Electrostatic potential on the protein-binding site is one of the properties regulating protein-protein interactions. In this study, we investigated the electrostatic potential on the interaction surface of capsid protein of HIV-2 strain GH123 and SIVmac239. Although HIV-2 GH123 and SIVmac239 capsid proteins share more than 87% amino acid identity, we observed a large difference between the two molecules with the HIV-2 GH123 molecule having predominantly positive and SIVmac239 predominantly negative electrostatic potential on the surface of the loop between α-helices 4 and 5 (L4/5). As L4/5 is one of the major determinants of Rh TRIM5α sensitivity of these viruses, the present results suggest that the binding site of the Rh TRIM5α may show complementarity to the HIV-2 GH123 capsid surface charge distribution.

The present study reported a new immunoblot assay, with revelation by R5- or X4-whole free human immunodeficiency virus (HIV) particles or recombinant gp160.

The main function attributed to the Rev proteins of immunodeficiency viruses is the shuttling of viral RNAs containing the Rev responsive element (RRE) via the CRM-1 export pathway from the nucleus to the cytoplasm. This restricts expression of structural proteins to the late phase of the lentiviral replication cycle. Using Rev-independent gag-pol expression plasmids of HIV-1 and simian immunodeficiency virus and lentiviral vector constructs, we have observed that HIV-1 and simian immunodeficiency virus Rev enhanced RNA encapsidation 20- to 70-fold, correlating well with the effect of Rev on vector titers. In contrast, cytoplasmic vector RNA levels were only marginally affected by Rev. Binding of Rev to the RRE or to a heterologous RNA element was required for Rev-mediated enhancement of RNA encapsidation. In addition to specific interactions of nucleocapsid with the packaging signal at the 5' end of the genome, the Rev/RRE system provides a second mechanism contributing to preferential encapsidation of genomic lentiviral RNA.

Human immunodeficiency virus (HIV) hides from the immune system in part by mimicking host antigens, including human leukocyte antigens. It is demonstrated here that HIV also mimics the V-β-D-J-β of approximately seventy percent of about 600 randomly selected human T cell receptors (TCR). This degree of mimicry is greater than any other human pathogen, commensal or symbiotic organism studied. These data suggest that HIV may be evolving into a commensal organism just as simian immunodeficiency virus has done in some types of monkeys. The gp120 envelope protein, Nef protein and Pol protein are particularly similar to host TCR, camouflaging HIV from the immune system and creating serious barriers to the development of safe HIV vaccines. One consequence of HIV mimicry of host TCR is that antibodies against HIV proteins have a significant probability of recognizing the corresponding TCR as antigenic targets, explaining the widespread observation of lymphocytotoxic autoantibodies in acquired immunodeficiency syndrome (AIDS). Quantitative enzyme-linked immunoadsorption assays (ELISA) demonstrated that every HIV antibody tested recognized at least one of twelve TCR, and as many as seven, with a binding constant in the 10-8 to 10-9 m range. HIV immunity also affects microbiome tolerance in ways that correlate with susceptibility to specific opportunistic infections.

In about half of patients infected with HIV-1 subtype B, viral populations shift from utilizing the transmembrane protein CCR5 to CXCR4, as well as or instead of CCR5, during late stage progression of the disease. How the relative adhesion efficiency and fusion competency of the viral Env proteins relate to infection during this transition is not well understood. Using a virus-cell fusion assay and live-cell single-molecule force spectroscopy, we compare the entry competency of viral clones to tensile strengths of the individual Env-receptor bonds of Env proteins obtained from a HIV-1 infected patient prior to and during coreceptor switching. The results suggest that the genetic determinants of viral entry were predominantly enriched in the C3, HR1 and CD regions rather than V3. Env proteins can better mediate entry into cells after coreceptor switch; this effective entry capacity does not correlate with the bond strengths between viral Env and cellular receptors.

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.

During infection, the human immunodeficiency virus type 1 (HIV-1) manipulates host cell mechanisms to its advantage, thereby controlling its replication or latency, and evading immune responses. Sumoylation is an essential post-translational modification that controls vital cellular activities including proliferation, stemness, or anti-viral immunity. SUMO peptides oppose pathogen replication and mediate interferon-dependent anti-viral activities. In turn, several viruses and bacteria attack sumoylation to disarm host immune responses. Here, we show that HIV-1 impairs cellular sumoylation and targets the host SUMO E1-activating enzyme. HIV-1 expression in cultured HEK293 cells or in CD4+ Jurkat T lymphocytes diminishes sumoylation by both SUMO paralogs, SUMO1 and SUMO2/3. HIV-1 causes a sharp and specific decline in UBA2 protein levels, a subunit of the heterodimeric SUMO E1 enzyme, which likely serves to reduce the efficiency of global protein sumoylation. Furthermore, HIV-1-infected individuals display a significant reduction in total leukocyte sumoylation that is uncoupled from HIV-induced cytopenia. Because sumoylation is vital for immune function, T-cell expansion and activity, loss of sumoylation during HIV disease may contribute to immune system deterioration in patients.

Unintegrated HIV-1 DNA serves as transcriptionally active templates in HIV-infected cells. Several host factors including NF-κβ enhance HIV-1 transcription. HIV-1 induced NF-κβ activation can be suppressed by viral protein U (Vpu). Interestingly HIV-1 Vpu shares amino acid homology with cellular Twik-related Acid Sensitive K+ (TASK) channel 1 and the proteins physically interact in cultured cells and AIDS lymphoid tissue. Furthermore, the first transmembrane domain of TASK-1 is functionally interchangeable with Vpu and like Vpu enhances HIV-1 release.

Understanding the molecular mechanisms involved in the assembly of viruses is essential for discerning how viruses transmit from cell to cell and host to host. Although molecular aspects of assembly have been studied for many viruses, we still have little information about these events in real time. Enveloped viruses such as HIV that assemble at, and bud from, the plasma membrane have been studied in some detail using live cell fluorescence imaging techniques; however, these approaches provide little information about the real-time morphological changes that take place as viral components come together to form individual virus particles. Here we used correlative scanning ion conductance microscopy and fluorescence confocal microscopy to measure the topological changes, together with the recruitment of fluorescently labeled viral proteins such as Gag and Vpr, during the assembly and release of individual HIV virus-like particles (VLPs) from the top, nonadherent surfaces of living cells. We show that 1) labeling of viral proteins with green fluorescent protein affects particle formation, 2) the kinetics of particle assembly on different plasma membrane domains can vary, possibly as a consequence of differences in membrane biophysical properties, and 3) VLPs budding from the top, unimpeded surface of cells can reach full size in 20 s and disappear from the budding site in 0.5 to 3 min from the moment curvature is initially detected, significantly faster than has been previously reported.

After cell entry, HIV undergoes rapid transport toward the nucleus using microtubules and microfilaments. Neither the cellular cytoplasmic components nor the viral proteins that interact to mediate transport have yet been identified. Using a yeast two-hybrid screen, we identified four cytoskeletal components as putative interaction partners for HIV-1 p24 capsid protein: MAP1A, MAP1S, CKAP1, and WIRE. Depletion of MAP1A/MAP1S in indicator cell lines and primary human macrophages led to a profound reduction in HIV-1 infectivity as a result of impaired retrograde trafficking, demonstrated by a characteristic accumulation of capsids away from the nuclear membrane, and an overall defect in nuclear import. MAP1A/MAP1S did not impact microtubule network integrity or cell morphology but contributed to microtubule stabilization, which was shown previously to facilitate infection. In addition, we found that MAP1 proteins interact with HIV-1 cores both in vitro and in infected cells and that interaction involves MAP1 light chain LC2. Depletion of MAP1 proteins reduced the association of HIV-1 capsids with both dynamic and stable microtubules, suggesting that MAP1 proteins help tether incoming viral capsids to the microtubular network, thus promoting cytoplasmic trafficking. This work shows for the first time that following entry into target cells, HIV-1 interacts with the cytoskeleton via its p24 capsid protein. Moreover, our results support a role for MAP1 proteins in promoting efficient retrograde trafficking of HIV-1 by stimulating the formation of stable microtubules and mediating the association of HIV-1 cores with microtubules.

This chapter discusses human immunodeficiency virus type 1 (HIV-1) associated with CD4 downmodulation. It also discusses the structure and function of CD4 and p56lck and factors involved in hiv-1-associated cd4 downmodulation. There are, at present, at least three HIV-1 gene products known to be involved in cell surface CD4 downmodulation. These are Nef, Vpu, and gp160. Whereas Nef is expressed during the early phase of HIV-1 gene expression, both Vpu and gp160, which appear to act coordinately, are expressed during the late phase. This functional convergence of HIV-1 proteins on cell surface CD4 downmodulation, whether specific or nonspecific in activity, suggests that this event is of critical importance in the life cycle of HIV-1. Further elucidation of the mechanisms that underlie CD4 cell surface downmodulation may lead to the development of novel strategies aimed at preventing such events, and potentially to the development of new therapeutic approaches.

As the hosts of lentiviruses, almost 40 species of felids (family Felidae) are distributed around the world, and more than 20 feline species test positive for feline immunodeficiency virus (FIV), a lineage of lentiviruses. These observations suggest that FIVs globally infected a variety of feline species through multiple cross-species transmission events during a million-year history. Cellular restriction factors potentially inhibit lentiviral replication and limit cross-species lentiviral transmission, and cellular APOBEC3 deaminases are known as a potent restriction factor. In contrast, lentiviruses have evolutionary-acquired viral infectivity factor (Vif) to neutralize the APOBEC3-mediated antiviral effect. Because the APOBEC3-Vif interaction is strictly specific for viruses and their hosts, a comprehensive investigation focusing on Vif-APOBEC3 interplay can provide clues that will elucidate the roles of this virus-host interplay on cross-species transmission of lentiviruses. Here, we performed a comprehensive investigation with 144 patterns of a round robin test using 18 feline APOBEC3Z3 genes, an antiviral APOBEC3 gene in felid, and 8 FIV Vifs and derived a matrix showing the interplay between feline APOBEC3Z3 and FIV Vif. We particularly focused on the interplay between the APOBEC3Z3 of three felids (domestic cat, ocelot, and Asian golden cat) and an FIV Vif (strain Petaluma), and revealed that residues 65 and 66 of the APOBEC3Z3 protein of multiple felids are responsible for the counteraction triggered by FIV Petaluma Vif. Altogether, our findings can be a clue to elucidate not only the scenarios of the cross-species transmissions of FIVs in felids but also the evolutionary interaction between mammals and lentiviruses. IMPORTANCE Most of the emergences of new virus infections originate from the cross-species transmission of viruses. The fact that some virus infections are strictly specific for the host species indicates that certain "species barriers" in the hosts restrict cross-species jump of viruses, while viruses have evolutionary acquired their own "arms" to overcome/antagonize/neutralize these hurdles. Therefore, understanding of the molecular mechanism leading to successful cross-species viral transmission is crucial for considering the menus of the emergence of novel pathogenic viruses. In the field of retrovirology, APOBEC3-Vif interaction is a well-studied example of the battles between hosts and viruses. Here, we determined the sequences of 11 novel feline APOBEC3Z3 genes and demonstrated that all 18 different feline APOBEC3Z3 proteins tested exhibit anti-feline immunodeficiency virus (FIV) activity. Our comprehensive investigation focusing on the interplay between feline APOBEC3 and FIV Vif can be a clue to elucidate the scenarios of the cross-species transmissions of FIVs in felids.

The safety and immunogenicity of candidate human immunodeficiency virus type 1 (HIV-1) vaccines have been studied in > 1500 healthy, seronegative (HIV-1-uninfected) subjects. HIV-1 envelope proteins, gp160 and gp120, have been the most extensively investigated. A live virus vector construct, vaccinia with insertion of the HIV-1 env gene, has also been studied. HIV-1 candidate vaccines have been well tolerated, with no acute or longer-term serious toxicity. Intramuscular multidose gp120 vaccines induce neutralizing antibodies, lymphoproliferative responses, and anti-HIV-1 CD4 cytotoxic T cell (CTL) activity. Immunization with the vaccinia-env construct, followed by a boost with an envelope protein, also induces neutralizing antibodies, and anti-HIV-1 CTL activity (CD8, major histocompatibility complex class I-restricted) has been observed. To date, serum from vaccinees can neutralize laboratory-adapted HIV-1 strains in vitro but not primary isolates; the significance of this observation is unknown. Additional approaches to vaccination against HIV-1 are in development.

RT-SHIV is a chimera of simian immunodeficiency virus (SIV) containing the reverse transcriptase (RT)-encoding region of human immunodeficiency virus type 1 (HIV-1) within the backbone of SIVmac239. It has been used in a non-human primate model for studies of non-nucleoside RT inhibitors (NNRTI) and highly active antiretroviral therapy (HAART). We and others have identified several mutations that arise in the "foreign" HIV-1 RT of RT-SHIV during in vivo replication. In this study we catalogued amino acid substitutions in the HIV-1 RT and in regions of the SIV backbone with which RT interacts that emerged 30 weeks post-infection from seven RT-SHIV-infected rhesus macaques. The virus set points varied from relatively high virus load, moderate virus load, to undetectable virus load. The G196R substitution in RT was detected from 6 of 7 animals at week 4 post-infection and remained in virus from 4 of 6 animals at week 30. Virus from four high virus load animals showed several common mutations within RT, including L74V or V75L, G196R, L214F, and K275R. The foreign RT from high virus load isolates exhibited as much variation as that of the highly variable envelope surface glycoprotein, and 10-fold higher than that of the native RT of SIVmac239. Isolates from moderate virus load animals showed much less variation in the foreign RT than the high virus load isolates. No variation was found in SIVmac239 genes known to interact with RT. Our results demonstrate substantial adaptation of the foreign HIV-1 RT in RT-SHIV-infected macaques, which most likely reflects selective pressure upon the foreign RT to attain optimal activity within the context of the chimeric RT-SHIV and the rhesus macaque host.

We previously developed CCR5-tropic neutralization-resistant simian/human immunodeficiency virus (SHIV) strains and a rhesus macaque model of infection with these SHIVs. We induced the production of neutralizing antibodies (nAbs) against HIV-1 by infecting rhesus macaques with different neutralization-resistant SHIV strains. First, SHIV-MK1 (MK1) (neutralization susceptible, tier 1B) with CCR5 tropism was generated from SHIV-KS661 using CXCR4 as the main co-receptor. nAbs against parental-lineage and heterologous tier 2 viruses were induced by tier 1B virus (MK1) infection of the rhesus macaque MM482. We analyzed viral resistance to neutralization over time in MM482 and observed that the infecting virus mutated from tier 1B to tier 2 at 36 weeks postinfection (wpi). In addition, an analysis of mutations showed that N169D, K187E, S190N, S239, T459N (T459D at 91 wpi), and V842A mutations were present after 36 wpi. This led to the appearance of neutralization-resistant viral clones. In addition, MK1 was passaged in three rhesus macaques to generate neutralization-resistant SHIV-MK38 (MK38) (tier 2). We evaluated nAb production by rhesus macaques infected with SHIV-MK38 #818 (#818) (tier 2), a molecular clone of MK38. Neutralization of the parental lineage was induced earlier than in macaques infected with tier 1B virus, and neutralization activity against heterologous tier 2 virus was beginning to develop. Therefore, CCR5-tropic neutralization-resistant SHIV-infected rhesus macaques may be useful models of anti-HIV-1 nAb production and will facilitate the development of a vaccine that elicits nAbs against HIV-1.

The 'Human Immunodeficiency Virus Type 1 (HIV-1), Human Protein Interaction Database', available through the National Library of Medicine at www.ncbi.nlm.nih.gov/RefSeq/HIVInteractions, was created to catalog all interactions between HIV-1 and human proteins published in the peer-reviewed literature. The database serves the scientific community exploring the discovery of novel HIV vaccine candidates and therapeutic targets. To facilitate this discovery approach, the following information for each HIV-1 human protein interaction is provided and can be retrieved without restriction by web-based downloads and ftp protocols: Reference Sequence (RefSeq) protein accession numbers, Entrez Gene identification numbers, brief descriptions of the interactions, searchable keywords for interactions and PubMed identification numbers (PMIDs) of journal articles describing the interactions. Currently, 2589 unique HIV-1 to human protein interactions and 5135 brief descriptions of the interactions, with a total of 14,312 PMID references to the original articles reporting the interactions, are stored in this growing database. In addition, all protein-protein interactions documented in the database are integrated into Entrez Gene records and listed in the 'HIV-1 protein interactions' section of Entrez Gene reports. The database is also tightly linked to other databases through Entrez Gene, enabling users to search for an abundance of information related to HIV pathogenesis and replication.

Human immunodeficiency virus-1 (HIV) infection of the central nervous system promotes neuronal injury that culminates in HIV-associated neurocognitive disorders. Viral proteins, including transactivator of transcription (Tat), have emerged as leading candidates to explain HIV-mediated neurotoxicity, though the mechanisms remain unclear. Tat transgenic mice or neurons exposed to Tat, which show neuronal loss, exhibit smaller mitochondria as compared to controls. To provide an experimental clue as to which mechanisms are used by Tat to promote changes in mitochondrial morphology, rat cortical neurons were exposed to Tat (100 nM) for various time points. Within 30 min, Tat caused a significant reduction in mitochondrial membrane potential, a process that is regulated by fusion and fission. To further assess whether Tat changes these processes, fission and fusion proteins dynamin-related protein 1 (Drp1) and mitofusin-2 (Mfn2), respectively, were measured. We found that Drp1 levels increased beginning at 2 h after Tat exposure while Mfn2 remained unchanged. Moreover, increased levels of an active form of Drp1 were found to be present following Tat exposure. Furthermore, Drp1 and calcineurin inhibitors prevented Tat-mediated effects on mitochondria size. These findings indicate that mitochondrial fission is likely the leading factor in Tat-mediated alterations to mitochondrial morphology. This disruption in mitochondria homeostasis may contribute to the instability of the organelle and ultimately neuronal cell death following Tat exposure.

Background.  The epidemic of sexually transmitted hepatitis C virus (HCV) infection among human immunodeficiency virus (HIV)-infected men who have sex with men (MSM) has been documented for over a decade. Despite this, there is no consensus as to the risk factors for sexual acquisition of HCV in these men. Methods.  We obtained paired semen and blood samples at 2-week intervals from HIV-infected MSM with recent and chronic HCV infection and quantified HCV in semen. Results.  Hepatitis C virus was quantified in 59 semen specimens from 33 men. Hepatitis C virus was shed in 16 (27%) of semen specimens from 11 (33%) of the men. Median HCV viral load (VL) in semen was 1.49 log10 IU/mL. Hepatitis C virus VL in blood was significantly higher at the time of HCV shedding in semen than when HCV shedding in semen was not detected (P = .002). Furthermore, there was a significant correlation between the HCV VL in blood and semen overall (rs = 0.41; P = .001), and in the subgroup with recent HCV infection (rs = 0.37; P = .02), but not in the subgroup with chronic HCV infection (rs = 0.34; P = .1). Conclusions.  One third of HIV-infected MSM coinfected with HCV shed HCV into their semen. Based on the HCV VL in semen in this study, an average ejaculate would deliver up to 6630 IU of virus into the rectum of the receptive partner. Therefore, our data strongly support that condoms should be used during anal intercourse among MSM to prevent transmission of HCV.

The targets for licensed drugs used for the treatment of human immunodeficiency virus type 1 (HIV-1) are confined to the viral reverse transcriptase (RT), protease (PR), and the gp41 transmembrane protein (TM). While currently approved drugs are effective in controlling HIV-1 infections, new drug targets and agents are needed due to the eventual emergence of drug resistant strains and drug toxicity. Our increased understanding of the virus life-cycle and how the virus interacts with the host cell has unveiled novel mechanisms for blocking HIV-1 replication. This review focuses on inhibitors that target the late stages of virus replication including the synthesis and trafficking of the viral polyproteins, viral assembly, maturation and budding. Novel approaches to blocking the oligomerization of viral enzymes and the interactions between viral proteins and host cell factors, including their feasibility as drug targets, are discussed.

Expression of the feline immunodeficiency virus (FIV) accessory protein OrfA (or Orf2) is critical for efficient viral replication in lymphocytes, both in vitro and in vivo. OrfA has been reported to exhibit functions in common with the human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) accessory proteins Vpr and Tat, although the function of OrfA has not been fully explained. Here, we use microarray analysis to characterize how OrfA modulates the gene expression profile of T-lymphocytes. The primary IL-2-dependent T-cell line 104-C1 was transduced to express OrfA. Functional expression of OrfA was demonstrated by trans complementation of the OrfA-defective clone, FIV-34TF10. OrfA-expressing cells had a slightly reduced cell proliferation rate but did not exhibit any significant alteration in cell cycle distribution. Reverse-transcribed RNA from cells expressing green fluorescent protein (GFP) or GFP+OrfA were hybridized to Affymetrix HU133 Plus 2.0 microarray chips representing more than 47,000 genome-wide transcripts. By using two statistical approaches, 461 (Rank Products) and 277 (ANOVA) genes were identified as modulated by OrfA expression. The functional relevance of the differentially expressed genes was explored by Ingenuity Pathway Analysis. The analyses revealed alterations in genes critical for RNA post-transcriptional modifications and protein ubiquitination as the two most significant functional outcomes of OrfA expression. In these two groups, several subunits of the spliceosome, cellular splicing factors and family members of the proteasome-ubiquitination system were identified. These findings provide novel information on the versatile function of OrfA during FIV infection and indicate a fine-tuning mechanism of the cellular environment by OrfA to facilitate efficient FIV replication.