HIV-1 Gag Expression Research Articles

HTLV-2 Encoded Antisense Protein APH-2 Suppresses HIV-1 Replication.

Antisense protein of Human T-cell Leukemia Virus Type 2 (HTLV-2), also called APH-2, negatively regulates the HTLV-2 and helps the virus to maintain latency via scheming the transcription. Despite the remarkable occurrence of HTLV-2/HIV-1 co-infection, the role of APH-2 influencing HIV-1 replication kinetics is poorly understood and needs investigation. In this study, we investigated the plausible role of APH-2 regulating HIV-1 replication. Herein, we report that the overexpression of APH-2 not only hampered the release of HIV-1 pNL4.3 from 293T cells in a dose-dependent manner but also affected the cellular gag expression. A similar and consistent effect of APH-2 overexpression was also observed in case of HIV-1 gag expression vector HXB2 pGag-EGFP. APH-2 overexpression also inhibited the ability of HIV-1 Tat to transactivate the HIV-1 LTR-driven expression of luciferase. Furthermore, the introduction of mutations in the IXXLL motif at the N-terminal domain of APH-2 reverted the inhibitory effect on HIV-1 Tat-mediated transcription, suggesting the possible role of this motif towards the downregulation of Tat-mediated transactivation. Overall, these findings indicate that the HTLV-2 APH-2 may affect the HIV-1 replication at multiple levels by (a) inhibiting the Tat-mediated transactivation and (b) hampering the virus release by affecting the cellular gag expression.

Helicobacter pylori Reactivates Human Immunodeficiency Virus-1 in Latently Infected Monocytes with Increased Expression of IL-1β and CXCL8.

BackgroundHelicobacter pylori are gram-negative bacteria, which colonize the human stomach. More than 50% of the world’s population is infected by H. pylori. Based on the high prevalence of H. pylori, it is very likely that HIV and H. pylori infection may coexist. However, the molecular events that occur during HIV-H. pylori co-infection remain unclear. Latent HIV reservoirs are the major obstacle in HIV cure despite effective therapy. Here, we explored the effect of H. pylori stimulation on latently HIV-infected monocytic cell line U1.Methods High throughput RNA-Seq using Illumina platform was performed to analyse the change in transcriptome between unstimulated and H. pylori-stimulated latently HIV-infected U1 cells. Transcriptome analysis identified potential genes and pathways involved in the reversal of HIV latency using bioinformatic tools that were validated by real-time PCR.ResultsH. pylori stimulation increased the expression of HIV-1 Gag, both at transcription (p<0.001) and protein level. H. pylori stimulation also increased the expression of proinflammatory cytokines IL-1β, CXCL8 and CXCL10 (p<0.0001). Heat-killed H. pylori retained their ability to induce HIV transcription. RNA-Seq analysis revealed 197 significantly upregulated and 101 significantly downregulated genes in H. pylori-stimulated U1 cells. IL-1β and CXCL8 were found to be significantly upregulated using transcriptome analysis, which was consistent with real-time PCR data.ConclusionH. pylori reactivate HIV-1 in latently infected monocytes with the upregulation of IL-1β and CXCL8, which are prominent cytokines involved in the majority of inflammatory pathways. Our results warrant future in vivo studies elucidating the effect of H. pylori in HIV latency and pathogenesis.

Screening for small molecule inhibitors of HIV-1 Gag expression.

The control of RNA processing plays an important role in the nature and quantity of protein generated from mammalian genes. Consequently, efforts to manipulate RNA processing have the capacity to significantly impact gene function. Although multiple strategies have been developed to alter splice site selection using oligonucleotide occlusion of splice sites or splicing regulatory elements, systemic delivery of such agents remains problematic. Outlined in this chapter is a protocol to screen for small molecule inhibitors of HIV-1 Gag expression that have been subsequently determined to modulate viral RNA processing. Identification and characterization of such RNA processing modulators offers the potential for the development of therapeutic lead compounds or probes for investigating the mechanism underlying the regulation of select RNA processing events.

Identification of small molecule modulators of HIV-1 Tat and Rev protein accumulation

BackgroundHIV-1 replication is critically dependent upon controlled processing of its RNA and the activities provided by its encoded regulatory factors Tat and Rev. A screen of small molecule modulators of RNA processing identified several which inhibited virus gene expression, affecting both relative abundance of specific HIV-1 RNAs and the levels of Tat and Rev proteins.ResultsThe screen for small molecules modulators of HIV-1 gene expression at the post-transcriptional level identified three (a pyrimidin-7-amine, biphenylcarboxamide, and benzohydrazide, designated 791, 833, and 892, respectively) that not only reduce expression of HIV-1 Gag and Env and alter the accumulation of viral RNAs, but also dramatically decrease Tat and Rev levels. Analyses of viral RNA levels by qRTPCR and RTPCR indicated that the loss of either protein could not be attributed to changes in abundance of the mRNAs encoding these factors. However, addition of the proteasome inhibitor MG132 did result in significant restoration of Tat expression, indicating that the compounds are affecting Tat synthesis and/or degradation. Tests in the context of replicating HIV-1 in PBMCs confirmed that 791 significantly reduced virus replication. Parallel analyses of the effect of the compounds on host gene expression revealed only minor changes in either mRNA abundance or alternative splicing. Subsequent tests suggest that 791 may function by reducing levels of the Tat/Rev chaperone Nap1.ConclusionsThe three compounds examined (791, 833, 892), despite their lack of structural similarity, all suppressed HIV-1 gene expression by preventing accumulation of two key HIV-1 regulatory factors, Tat and Rev. These findings demonstrate that selective disruption of HIV-1 gene expression can be achieved.

Expression of HIV-1 gag and env genes using the vesicular stomatitis virus vector system

Traditional vaccine methods have long been employed to control widespread infectious diseases, but so far, all commercially available vaccine strategies have been inadequate in efforts to develop an effective therapeutic HIV vaccine. However, recent advancements in immunological research have led to the generation of novel vaccine strategies, one of which is the recombinant virus vaccine, a method of particular interest that has shown promise in the clearance of HIV infection within HIV-positive patients who have retained immunocompetence. This study examined the stability of expression of HIV-1 genes, gag and env, through a recombinant virus vector, a recombinant vesicular stomatitis virus (VSV), a temperature-sensitive mutant genetically modified to contain the select HIV-1 genes (VSVInd(GML)HIV-1gag-env). For SDS-PAGE, cells infected with VSVInd(GML) temperature-sensitive mutants were incubated at 31 °C and 37 °C, the permissible and semi-permissible growth conditions respectively. Western blot analyses were used to quantitate levels of protein expression of full VSV proteins, Gag, and Env using a primary rabbit antibody of anti-VSV anti-serum and a secondary anti-IgG from rabbit, a primary antibody of anti-p24 anti-serum and a secondary anti-IgG from rabbit, and a primary goat antibody, anti-gp120, and a secondary anti-IgG from goat, respectively. Results indicated that the VSVInd(GML) vector system allowed for high levels of expression of HIV-1 gag and env genes. It is known that the expression of these genes induce the production of major neutralizing antibodies and the stimulation of cytotoxic T lymphocytes, therefore this finding reveals the potential to use a genetically modified recombinant VSV as a universal vector for the development of recombinant virus vaccines. Specifically, the VSVInd(GML) mutant vector is thus an attractive candidate for the viral vector of a therapeutic HIV vaccine system.

103: Identification of a novel interferon-stimulated gene whose product significantly restricts retrovirus replication

Interferons confer cells resistant to retrovirus infection by inducing anti-viral cellular factors, such as tetherin, APOBEC3G, and Mx2. It is believed that many factors inhibiting viral infections among interferon-stimulated genes (ISGs) remain to be identified. On the other hand, we found efficient restriction of retrovirus infection by a compound that inhibits S–S bond formation at acidic pH. Thus, it was postulated that an acidic endosome-localized, S–S bond digesting factor among ISGs may restrict retrovirus replication. We here report for the first time that such a cellular factor efficiently inhibits MLV and HIV-1 replication. When the factor was expressed in target cells, transduction efficiency of HIV-1 vector pseudotyped with ecotropic, amphotropic, xenotropic MLV, or VSV was significantly reduced. In addition, when HIV-1 vector-producing cells were transfected with the factor, HIV-1 Gag expression and virion production were significantly suppressed. The factor digested S–S bonds of viral envelope proteins and of CD63, which is involved in HIV-1 virion release. A CD63 mutant containing amino acid substitutions at conserved cysteine residues in the second extracellular loop suppressed virion release like GILT. Interferon treatment induced the factor expression, and its knockdown by shRNA abrogated interferon-induced inhibition of HIV-1 vector infection. These results indicate that interferon restricts HIV-1 entry and production at endosomal level by inducing the factor.

RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export.

Post-transcriptional control of HIV-1 Gag expression

Background The HIV-1 genomic RNA (gRNA) has several roles in the viral life cycle. In the nucleus, the gRNA is the pre-mRNA that is alternatively and incompletely spliced into mRNAs encoding Tat, Rev, Nef, Vif, Vpr, Vpu and Env. However, approximately 50% of the gRNA remains unspliced and is exported from the nucleus by the viral protein Rev using the CRM1 nuclear export pathway. In the cytoplasm, the gRNA serves as the mRNA for Gag and Gag-Pol and is also packaged into virions. As the gRNA is an unspliced, intron-containing mRNA with a long, highly structured 5’ UTR and rare codon usage in the open reading frames, how it interacts with cellular RNA metabolism and translation machinery to promote abundant Gag expression and infectious virion production remains unclear.

Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos

ObjectiveStudying the methylation status of long terminal repeats (LTR) and its relationship to gag expression of HIV-1 in order to explore regulation mechanism of HIV-1 gene expression in vertical transmission from sperm to embryo.Methods/Principal FindingsSperm samples were collected from a healthy donor and seven patients with HIV/AIDS. Zona-free hamster ova were fertilized by donor’s spermatozoa transfected with pIRES2-EGFP-LTR-gag and patient’s spermatozoa to obtain zygotes and 2-cell embryos, respectively. Interspecific in vitro fertilization, bisulfite sequencing PCR (BSP), RT-PCR, nested RT-PCR, nested real-time qRT-PCR and 2−△△Ct method, indirect immunofluoresence (IF) assay were performed. For donor’s samples, the methylation rates of HIV-1 LTR were 0.56%, 1.67%, 0.56%, 0.56% in plasmid, spermatozoa, zygotes and 2-cell embryos, respectively while spermatozoa were transfected with unmethylated plasmid, and were 95.0%, 84.44%, 3.3%, 1.67% while transfected with methylated plasmid. The positive bands for HIV-1 gag cDNA were detected in spermatozoa and 2-cell embryos. The positive signals for HIV-1 p24 Gag protein were detected in 2-cell embryos but not in spermatozoa. For patient’s samples, methylation rates of HIV-1 LTR were different in spermatozoa among patients. After fertilization, CpG sites in HIV-1 LTR were highly demethylated in zygotes and 2-cell embryos. The gag transcription levels increased with decreasing of methylation rates of HIV-1 LTR, which showed a strong negative correlations between gag transcription levels and methylation rates of HIV-LTR ether in the spermatozoa (r = −0.9877, P<0.0001) or in the sperm-derived 2-cell embryos (r = −0.9092, P = 0.0045).ConclusionLTR methylation regulates expression of HIV-1 gag in vertical transmission from sperm to embryo.

ESCRT-Independent Budding of HIV-1 Gag Virus-Like Particles from Saccharomyces cerevisiae Spheroplasts

Heterologous expression of HIV-1 Gag in a variety of host cells results in its packaging into virus-like particles (VLPs) that are subsequently released into the extracellular milieu. This phenomenon represents a useful tool for probing cellular factors required for viral budding and has contributed to the discovery of roles for ubiquitin ligases and the endosomal sorting complexes required for transport (ESCRTs) in viral budding. These factors are highly conserved throughout eukaryotes and have been studied extensively in the yeast Saccharomyces cerevisiae, a model eukaryote previously utilized as a host for the production of VLPs. We used heterologous expression of HIV Gag in yeast spheroplasts to examine the role of ESCRTs and associated factors (Rsp5, a HECT ubiquitin ligase of the Nedd4 family; Bro1, a homolog of Alix; and Vps4, the AAA-ATPase required for ESCRT function in all contexts/organisms investigated) in the generation of VLPs. Our data reveal: 1) characterized Gag-ESCRT interaction motifs (late domains) are not required for VLP budding, 2) loss of function alleles of the essential HECT ubiquitin ligase Rsp5 do not display defects in VLP formation, and 3) ESCRT function is not required for VLP formation from spheroplasts. These results suggest that the egress of HIV Gag from yeast cells is distinct from the most commonly described mode of exit from mammalian cells, instead mimicking ESCRT-independent VLP formation observed in a subset of mammalian cells. As such, budding of Gag from yeast cells appears to represent ESCRT-independent budding relevant to viral replication in at least some situations. Thus the myriad of genetic and biochemical tools available in the yeast system may be of utility in the study of this aspect of viral budding.

A facile quantitative assay for viral particle genesis reveals cooperativity in virion assembly and saturation of an antiviral protein

Conventional assays of viral particle assembly and release are time consuming and laborious. We have developed an enzymatic virus-like particle (VLP) genesis assay that rapid and quantitative and is also versatile and applicable to diverse viruses including HIV-1 and Ebola virus. Using this assay, which has a dynamic range of several orders of magnitude, we show that the efficiency of VLP assembly and release, i.e., the fraction of the expressed protein that is assembled into extracellular particles, is dependent on the absolute level of expression of either HIV-1 Gag or Ebola virus VP40. We also demonstrate that the activity of the antiviral factor tetherin is dependent on the level of HIV-1 Gag expression and the numbers of VLPs generated, and appears to become saturated as these parameters are increased.

Differential effects of hnRNP D/AUF1 isoforms on HIV-1 gene expression

Control of RNA processing plays a major role in HIV-1 gene expression. To explore the role of several hnRNP proteins in this process, we carried out a siRNA screen to examine the effect of depletion of hnRNPs A1, A2, D, H, I and K on HIV-1 gene expression. While loss of hnRNPs H, I or K had little effect, depletion of A1 and A2 increased expression of viral structural proteins. In contrast, reduced hnRNP D expression decreased synthesis of HIV-1 Gag and Env. Loss of hnRNP D induced no changes in viral RNA abundance but reduced the accumulation of HIV-1 unspliced and singly spliced RNAs in the cytoplasm. Subsequent analyses determined that hnRNP D underwent relocalization to the cytoplasm upon HIV-1 infection and was associated with Gag protein. Screening of the four isoforms of hnRNP D determined that, upon overexpression, they had differential effects on HIV-1 Gag expression, p45 and p42 isoforms increased viral Gag synthesis while p40 and p37 suppressed it. The differential effect of hnRNP D isoforms on HIV-1 expression suggests that their relative abundance could contribute to the permissiveness of cell types to replicate the virus, a hypothesis subsequently confirmed by selective depletion of p45 and p42.

Soluble multi-trimeric TNF superfamily ligand adjuvants enhance immune responses to a HIV-1 Gag DNA vaccine

DNA vaccines remain an important component of HIV vaccination strategies, typically as part of a prime/boost vaccination strategy with viral vector or protein boost. A number of DNA prime/viral vector boost vaccines are currently being evaluated for both preclinical studies and in Phase I and Phase II clinical trials. These vaccines would benefit from molecular adjuvants that increase correlates of immunity during the DNA prime. While HIV vaccine immune correlates are still not well defined, there are a number of immune assays that have been shown to correlate with protection from viral challenge including CD8+ T cell avidity, antigen-specific proliferation, and polyfunctional cytokine secretion. Recombinant DNA vaccine adjuvants composed of a fusion between Surfactant Protein D (SP-D) and either CD40 Ligand (CD40L) or GITR Ligand (GITRL) were previously shown to enhance HIV-1 Gag DNA vaccines. Here we show that similar fusion constructs composed of the TNF superfamily ligands (TNFSFL) 4-1BBL, OX40L, RANKL, LIGHT, CD70, and BAFF can also enhanced immune responses to a HIV-1 Gag DNA vaccine. BALB/c mice were vaccinated intramuscularly with plasmids expressing secreted Gag and SP-D-TNFSFL fusions. Initially, mice were analyzed 2 weeks or 7 weeks following vaccination to evaluate the relative efficacy of each SP-D-TNFSFL construct. All SP-D-TNFSFL constructs enhanced at least one Gag-specific immune response compared to the parent vaccine. Importantly, the constructs SP-D-4-1BBL, SP-D-OX40L, and SP-D-LIGHT enhanced CD8+ T cell avidity and CD8+/CD4+ T cell proliferation 7 weeks post vaccination. These avidity and proliferation data suggest that 4-1BBL, OX40L, and LIGHT fusion constructs may be particularly effective as vaccine adjuvants. Constructs SP-D-OX40L, SP-D-LIGHT, and SP-D-BAFF enhanced Gag-specific IL-2 secretion in memory T cells, suggesting these adjuvants can increase the number of self-renewing Gag-specific CD8+ and/or CD4+ T cells. Finally adjuvants SP-D-OX40L and SP-D-CD70 increased T(H)1 (IgG2a) but not T(H)2 (IgG1) antibody responses in the vaccinated animals. Surprisingly, the B cell-activating protein BAFF did not enhance anti-Gag antibody responses when given as an SP-D fusion adjuvant, but nonetheless enhanced CD4+ and CD8+ T cell responses. We present evidence that various SP-D-TNFSFL fusion constructs can enhance immune responses following DNA vaccination with HIV-1 Gag expression plasmid. These data support the continued evaluation of SP-D-TNFSFL fusion proteins as molecular adjuvants for DNA and/or viral vector vaccines. Constructs of particular interest included SP-D-OX40L, SP-D-4-1BBL, SP-D-LIGHT, and SP-D-CD70. SP-D-BAFF was surprisingly effective at enhancing T cell responses, despite its inability to enhance anti-Gag antibody secretion.

Translational Regulation of HIV-1 Replication by HIV-1 Rev Cellular Cofactors Sam68, eIF5A, hRIP, and DDX3

Nuclear export and translation of HIV-1 RNA are two important posttranscriptional events for HIV-1 gene expression and replication. HIV-1 Rev functions to export unspliced and incompletely spliced HIV-1 RNA from the nucleus to the cytoplasm; it requires interaction with several cellular cofactors such as Sam68, eIF5A, hRIP, and DDX3. Meanwhile, some studies have also implicated Rev and some of its cofactors such as Sam68 in HIV-1 RNA translation. Thus, in this study, we aimed to characterize the potential function of all these four Rev cofactors in HIV-1 RNA translation. Ectopic expression, siRNA knockdown, and trans-complementation assays confirmed that all these cofactors were very important for HIV-1 gene expression and production through Rev and, accordingly, Rev-dependent reporter gene expression. Importantly, these studies revealed for the first time that each of these cofactors also regulated Rev-independent reporter gene expression. To directly determine the roles of these cofactors in HIV-1 RNA translation, we designed and synthesized a full-length capped HIV-1 RNA in vitro, transfected it into cells to bypass the RNA nuclear export step, and determined HIV-1 Gag expression from the cytoplasmic RNA in the cells that had ectopically expressed or siRNA knocked down cofactors. Gag expression was found to closely correlate with the expression levels of all these cofactors. Furthermore, we took advantage of a HIV-1 internal ribosomal entry site (IRES)-based bicistronic reporter gene assay and determined the effects of these cofactors on cap-independent IRES-mediated HIV-1 translation. The results showed that DDX3, eIF5A, and hRIP enhanced HIV-1 IRES-mediated translation, whereas Sam68 did not. Taken together, these results show that HIV-1 Rev cofactors Sam68, eIF5A, hRIP, and DDX3 also function in the translation of HIV-1 RNA and suggest that the regulatory mechanisms of HIV-1 RNA translation are likely different among these cofactors.

A novel SR protein binding site in a cis-regulatory element of HIV-1

We have previously described a cis enhancing sequence (CES) in the gp41 region of the HIV-1 env gene. It could enhance HIV-1 Gag expression via both rev-dependent and CTE (constitutive transport element)-dependent pathways. We identified a functionally important and conserved region in the CES that contained a predicted binding sequence for an SR protein, ASF/SF2. We show here that ASF/SF2 bound to this sequence in an electrophoretic mobility shift assay and that the putative ASF/SF2-binding sequence was required for the enhancement of Gag expression by CES and might play a role in HIV-1 posttranscriptional regulation.

Japanese encephalitis virus-based replicon RNAs/particles as an expression system for HIV-1 Pr55 Gag that is capable of producing virus-like particles

Ectopic expression of the structural protein Pr55 Gag of HIV-1 has been limited by the presence of inhibitory sequences in the gag coding region that must normally be counteracted by HIV-1 Rev and RRE. Here, we describe a cytoplasmic RNA replicon based on the RNA genome of Japanese encephalitis virus (JEV) that is capable of expressing HIV-1 gag without requiring Rev/RRE. This replicon system was constructed by deleting all three JEV structural protein-coding regions (C, prM, and E) from the 5′-proximal region of the genome and simultaneously inserting an HIV-1 gag expression cassette driven by the internal ribosome entry site of encephalomyocarditis virus into the 3′-proximal noncoding region of the genome. Transfection of this JEV replicon RNA led to expression of Pr55 Gag in the absence of Rev/RRE in the cytoplasm of hamster BHK-21, human HeLa, and mouse NIH/3T3 cells. Production of the Pr55 Gag derived from this JEV replicon RNA appeared to be increased by ∼3-fold when compared to that based on an alphavirus replicon RNA. Biochemical and morphological analyses demonstrated that the Pr55 Gag proteins were released into the culture medium in the form of virus-like particles. We also observed that the JEV replicon RNAs expressing the Pr55 Gag could be encapsidated into single-round infectious JEV replicon particles when transfected into a stable packaging cell line that provided the three JEV structural proteins in trans. This ectopic expression of the HIV-1 Pr55 Gag by JEV-based replicon RNAs/particles in diverse cell types may represent a useful molecular platform for various biological applications in medicine and industry.

Efficient production of HIV-1 viral-like particles in mouse cells

Previous efforts to develop a mouse model for HIV/AIDS have been impaired by multiple blocks to HIV replication, including barriers to viral entry, proviral transcription, and assembly. Expression of human cofactors in murine cells overcomes early restrictions, but does not lead to the production of infectious HIV particles. Here we show that stable expression of a codon-optimized synthetic HIV-1 Gag–Pol construct (sGP) in murine cell lines results in efficient Gag production and viral-like particle (VLP) release. Stable expression of the sGP construct in murine cells such as NIH3T3 and A9 improved Gag processing resulting in efficient VLP release comparable to that found in human cells. Using highly efficient transient transfection procedures, we increased Gag expression, and were able to produce infectious HIV particles in NIH3T3 cells. However, the infectivity of VLPs produced in murine cells was significantly below that generated in 293T cells. Reduced infectivity of VLPs produced in murine cells correlated with lower HIV reporter RNA levels in these cells. Taken together, improving the expression of HIV-1 Gag–Pol by using the sGP construct overcomes, at least in part, late restrictions in murine cells.

Differential control of CXCR4 and CD4 downregulation by HIV-1 Gag

BackgroundThe ESCRT (endosomal sorting complex required for transport) machinery functions to sort cellular receptors into the lumen of the multivesicular body (MVB) prior to lysosomal degradation. ESCRT components can also be recruited by enveloped viruses to sites of viral assembly where they have been proposed to mediate viral egress. For example, HIV-1 budding is dependent on Gag-mediated recruitment of the cellular ESCRTs-I, -III, AIP1/Alix and Vps4 proteins. Viral recruitment of ESCRT proteins could therefore impact on host cell processes such as receptor downregulation.ResultsHere we show that downregulation of the HIV-1 co-receptor, CXCR4, by its ligand SDF-1, is ESCRT-I dependent. Expression of HIV-1 Gag attenuated downregulation of CXCR4, resulting in accumulation of undegraded receptors within intracellular compartments. The effect of Gag was dependent on an ESCRT-I interacting motif within the C-terminal p6 region of Gag. In contrast, PMA-induced downregulation of the HIV-1 receptor CD4 was independent of ESCRT-I and Vps4; HIV-1 Gag had no effect on this process.ConclusionThese results establish that the HIV-1 receptor, CD4, and co-receptor, CXCR4 are differentially regulated by ESCRT proteins. HIV-1 Gag selectively modulates protein sorting at the MVB, interfering with ESCRT-I dependent but not ESCRT-I independent processes.

A quasi-lentiviral green fluorescent protein reporter exhibits nuclear export features of late human immunodeficiency virus type 1 transcripts

We have previously shown that Rev-dependent expression of HIV-1 Gag from CMV immediate early promoter critically depends on the AU-rich codon bias of the gag gene. Here, we demonstrate that adaptation of the green fluorescent protein (GFP) reporter gene to HIV codon bias is sufficient to turn this hivGFP RNA into a quasi-lentiviral message following the rules of late lentiviral gene expression. Accordingly, GFP expression was significantly decreased in transfected cells strictly correlating with reduced RNA levels. In the presence of the HIV 5′ major splice donor, the hivGFP RNAs were stabilized in the nucleus and efficiently exported to the cytoplasm following fusion of the 3′ Rev-responsive element (RRE) and coexpression of HIV-1 Rev. This Rev-dependent translocation was specifically inhibited by leptomycin B suggesting export via the CRM1-dependent pathway used by late lentiviral transcripts. In conclusion, this quasi-lentiviral reporter system may provide a new platform for developing sensitive Rev screening assays.

HIV-1 and MLV Gag proteins are sufficient to recruit APOBEC3G into virus-like particles

The cytidine deaminase hAPOBEC3G is an antiviral human factor that counteracts the replication of HIV-1 in absence of the Vif protein. hAPOBEC3G is packaged into virus particles and lethally hypermutates HIV-1. In this work, we examine the mechanisms governing hAPOBEC3G packaging. By GST pull-down and co-immunoprecipitation assays, we show that hAPOBEC3G binds to HIV-1 Pr55 Gag and its NC domain and to the RT and IN domains contained in Pr160 Gag-Pol. We demonstrate that the expression of HIV-1 Gag is sufficient to induce the packaging of hAPOBEC3G into Gag particles. Gag-Pol polypeptides containing RT and IN domains, as well as HIV-1 genomic RNA, seem not to be necessary for hAPOBEC3G packaging. Lastly, we show that hAPOBEC3G and its murine ortholog are packaged into HIV-1 and MLV Gag particles. We conclude that the Gag polypeptides from distant retroviruses have conserved domains allowing the packaging of the host antiviral factor APOBEC3G.