Human Immunodeficiency Virus Type 1 Gag Research Articles

HIV-1 Gag co-localizes with euchromatin histone marks at the nuclear periphery.

The traditional view of retrovirus assembly posits that packaging of gRNA by HIV-1 Gag occurs in the cytoplasm or at the plasma membrane. However, our previous studies showing that HIV-1 Gag enters the nucleus and binds to USvRNA at transcription sites suggest that gRNA selection may occur in the nucleus. In the present study, we observed that HIV-1 Gag trafficked to the nucleus and co-localized with USvRNA within 8 hours of expression. In infected T cells (J-Lat 10.6) reactivated from latency and in a HeLa cell line stably expressing an inducible Rev-dependent HIV-1 construct, we found that Gag preferentially localized with euchromatin histone marks associated with enhancer and promoter regions near the nuclear periphery, which is the favored site HIV-1 integration. These observations support the innovative hypothesis that HIV-1 Gag associates with euchromatin-associated histones to localize to active transcription sites, promoting capture of newly synthesized gRNA for packaging.

Interaction of TSG101 with the PTAP Motif in Distinct Locations of Gag Determines the Incorporation of HTLV-1 Env into the Retroviral Virion.

Human T-cell tropic virus type 1 (HTLV-1) is known to be mainly transmitted by cell-to-cell contact due to the lower infectivity of the cell-free virion. However, the reasons why cell-free HTLV-1 infection is poor remain unknown. In this study, we found that the retrovirus pseudotyped with HTLV-1 viral envelope glycoprotein (Env) was infectious when human immunodeficiency virus type 1 (HIV-1) was used to produce the virus. We found that the incorporation of HTLV-1 Env into virus-like particles (VLPs) was low when HTLV-1 Gag was used to produce VLPs, whereas VLPs produced using HIV-1 Gag efficiently incorporated HTLV-1 Env. The production of VLPs using Gag chimeras between HTLV-1 and HIV-1 Gag and deletion mutants of HIV-1 Gag showed that the p6 domain of HIV-1 Gag was responsible for the efficient incorporation of HTLV-1 Env into the VLPs. Further mutagenic analyses of the p6 domain of HIV-1 Gag revealed that the PTAP motif in the p6 domain of HIV-1 Gag facilitates the incorporation of HTLV-1 Env into VLPs. Since the PTAP motif is known to interact with tumor susceptibility gene 101 (TSG101) during the budding process, we evaluated the effect of TSG101 knockdown on the incorporation of HTLV-1 Env into VLPs. We found that TSG101 knockdown suppressed the incorporation of HTLV-1 Env into VLPs and decreased the infectivity of cell-free HIV-1 pseudotyped with HTLV-1 Env. Our results suggest that the interaction of TSG101 with the PTAP motif of the retroviral L domain is involved not only in the budding process but also in the efficient incorporation of HTLV-1 Env into the cell-free virus.

HIV-1 Gag MA domain binds to cardiolipin in a binding mode distinct from virus assemble mediator PI(4,5)P2.

The human immunodeficiency virus type 1 (HIV-1) Gag protein is responsible for facilitating HIV-1 virion assembly and budding. Our study demonstrates that cardiolipin (CL), a component found in the inner mitochondrial membrane, exhibits the highest binding affinity to the N-terminal MA domain of the HIV-1 Gag protein within the lipid group of host cells. To assess this binding interaction, we synthesized short acyl chain derivatives of CL and employed surface plasmon resonance (SPR) analysis to determine the dissociation constants (Kd) for CL and the MA domain. Simultaneously, we examined the Kd of D-myo-phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) derivatives, known to play a crucial role in virion formation. Among all the derivatives, Tetra-C7 -CL exhibited the lowest Kd value (Kd = 30.8 ± 6.9 μM) for MA binding on the CL analog-immobilized sensorchip, indicating a higher affinity. Similarly, the Kd value of Di-C7 -PIP2 (Kd = 36.6 ± 4.7 μM) was the lowest on the PI(4,5)P2 analog-immobilized sensorchip. Thus, Tetra-C7 -CL binds to the MA domain using a distinct binding mode while displaying a comparable binding affinity to Di-C7 -PIP2. This discovery holds significant implications for comprehending the virological importance of CL-MA domain binding, such as its subcellular distribution, including mitochondrial translocation, and involvement in viral particle formation in concert with PI(4,5)P2 . Furthermore, this study has the potential to contribute to the development of drugs in the future.

HIV-2 Immature Particle Morphology Provides Insights into Gag Lattice Stability and Virus Maturation

Retrovirus immature particle morphology consists of a membrane enclosed, pleomorphic, spherical and incomplete lattice of Gag hexamers. Previously, we demonstrated that human immunodeficiency virus type 2 (HIV-2) immature particles possess a distinct and extensive Gag lattice morphology. To better understand the nature of the continuously curved hexagonal Gag lattice, we have used the single particle cryo-electron microscopy method to determine the HIV-2 Gag lattice structure for immature virions. The reconstruction map at 5.5 Å resolution revealed a stable, wineglass-shaped Gag hexamer structure with structural features consistent with other lentiviral immature Gag lattice structures. Cryo-electron tomography provided evidence for nearly complete ordered Gag lattice structures in HIV-2 immature particles. We also solved a 1.98 Å resolution crystal structure of the carboxyl-terminal domain (CTD) of the HIV-2 capsid (CA) protein that identified a structured helix 12 supported via an interaction of helix 10 in the absence of the SP1 region of Gag. Residues at the helix 10–12 interface proved critical in maintaining HIV-2 particle release and infectivity. Taken together, our findings provide the first 3D organization of HIV-2 immature Gag lattice and important insights into both HIV Gag lattice stabilization and virus maturation.

Subtype Distribution and Drug Resistance Patterns Among HIV-1 Strains Prevalent in Makassar, Indonesia.

Human immunodeficiency virus type 1 (HIV-1) is characterized by a large degree of genetic variability because of high rates of recombination and mutation, sizable population sizes, and rapid replication. Therefore, this study investigated HIV-1 subtype distribution and the appearance of drug resistance mutations (DRMs) in viruses that are prevalent in Makassar, South Sulawesi, Indonesia. The HIV-1 pol, env, and gag genes were amplified from 63 infected individuals and sequenced for a subtyping analysis. CRF01_AE was identified as the predominant HIV-1 circulating recombinant form (CRF) in Makassar, South Sulawesi, Indonesia. Subtype B and recombinant viruses containing CRF01_AE, CRF02_AG, and/or subtype B gene fragments were also detected. Several major DRMs against non-nucleoside reverse transcriptase inhibitors were found among antiretroviral therapy (ART)-experienced subjects, whereas ART-naive subjects did not possess any transmitted drug resistance. The prevalence of DRMs was very high among ART-experienced subjects; therefore, further surveillance is required in this region.

HIV-1 Gag Binds the Multi-Aminoacyl-tRNA Synthetase Complex via the EPRS Subunit.

Host factor tRNAs facilitate the replication of retroviruses such as human immunodeficiency virus type 1 (HIV-1). HIV-1 uses human tRNALys3 as the primer for reverse transcription, and the assembly of HIV-1 structural protein Gag at the plasma membrane (PM) is regulated by matrix (MA) domain-tRNA interactions. A large, dynamic multi-aminoacyl-tRNA synthetase complex (MSC) exists in the cytosol and consists of eight aminoacyl-tRNA synthetases (ARSs) and three other cellular proteins. Proteomic studies to identify HIV-host interactions have identified the MSC as part of the HIV-1 Gag and MA interactomes. Here, we confirmed that the MA domain of HIV-1 Gag forms a stable complex with the MSC, mapped the primary interaction site to the linker domain of bi-functional human glutamyl-prolyl-tRNA synthetase (EPRS), and showed that the MA-EPRS interaction was RNA dependent. MA mutations that significantly reduced the EPRS interaction reduced viral infectivity and mapped to MA residues that also interact with phosphatidylinositol-(4,5)-bisphosphate. Overexpression of EPRS or EPRS fragments did not affect susceptibility to HIV-1 infection, and knockdown of EPRS reduced both a control reporter gene and HIV-1 protein translation. EPRS knockdown resulted in decreased progeny virion production, but the decrease could not be attributed to selective effects on virus gene expression, and the specific infectivity of the virions remained unchanged. While the precise function of the Gag-EPRS interaction remains uncertain, we discuss possible effects of the interaction on either virus or host activities.

Probing P(T/S)XP peptides for novel structural interactions with Tsg101 UEV.

To complete their lifecycle, enveloped viruses such as human immunodeficiency virus type-1 (HIV-1) recruit host machinery to facilitate budding and egress. For HIV-1, this involves an interaction between a PTAP motif in the HIV-1 Gag polyprotein and the ubiquitin E2 variant (UEV) domain of tumor susceptibility gene 101 (Tsg101). The latter is part of the native endosomal sorting complex required for transport (ESCRT) machinery, which functions in endosomal cargo sorting but is hijacked by HIV-1 for plasma membrane budding. The interaction between the PTAP signal of HIV-1 Gag and the UEV domain of Tsg101 is well characterized biochemically and structurally, with the PTAP motif binding into a groove in the UEV domain. The HIV-1 PTAP sequence emulates similar P(T/S)XP signals that drive host protein interactions involved with ESCRT, where some host proteins may contain several potential P(T/S)XP motifs. This suggested potential competitive or cooperative action on the UEV surface, and so we screened two additional P(T/S)XP motifs from the ESCRT-0 protein Hrs and several motifs identified in other viruses for interaction with the Tsg101 UEV domain using solution nuclear magnetic resonance. In the process, we identified an additional interaction mode for P(T/S)XP peptides with the Tsg101 UEV, involving both a charged surface and a cluster of aromatic residues, neither of which are implicated in the previously characterized PTAP interaction. We identified several binding determinants of this P(T/S)XP interaction using alanine mutant peptides and combined chemical shift perturbations with distance data from paramagnetic relaxation enhancement experiments using spin-labeled peptides to build a structural model of the interaction on the UEV surface. This additional P(T/S)XP interaction may reflect a novel viral strategy to recruit Tsg101 and by extension ESCRT for egress, or hijacking of a different endogenous role of Tsg101 altogether.

Role of gRNA binding in HIV-1 Gag assembly process.

Assembly of human immunodeficiency virus type 1 (HIV-1) is a multi-step process that occurs at the host cell plasma membrane and is driven by a viral structural protein, Gag. Newly synthesized cytosolic Gag particles are targeted to the inner leaflet of the plasma membrane and membrane-bound Gag polyproteins interact with each other to form higher order multimers. Gag polyprotein is composed of multiple structured domains connected by flexible linkers. N-myristoylated matrix (MA) domain mediates membrane binding process, capsid (CA) and nucleocapsid (NC) domains play vital roles in Gag multimerization process. Interactions of gRNA with the zinc finger motifs of the NC domain facilitate gRNA packaging into virions. To investigate the effect of Gag-gRNA binding on the different multimerization states, we have performed all-atom molecular dynamics simulations of full-length Gag multimers (hexamer and hexamer-of-trimers) bound to an asymmetric membrane. MA domain multimerizes as a hexamer of trimers whereas cryo-ET data shows that CA domains are organized as hexameric lattice. The multidomain correlation of Gag polyprotein as well as the effect of multimerization of one domain to the dynamics of others are still not clear. Here we aim to gain atomic-level characterization of the inter and intra-protein interactions between different structural domains of Gag polyprotein in the presence/absence of the effective rigidity of NC domain, associated with gRNA binding. In the hexamer-of-trimers, the rigidity of NC domain is observed to modify the structure and dynamics of Gag monomers, resulting in a different binding surface for cytosolic Gag protein. In case of membrane-bound Gag hexamer, gRNA binding alters the dynamics of MA-CA linker domains, and we observe a signature of PIP2 and cholesterol enrichment in the membrane that can modify membrane binding affinity for Gag hexamer and facilitate recruitment of cytosolic Gag protein.

The HIV-1 Gag Protein Displays Extensive Functional and Structural Roles in Virus Replication and Infectivity.

Once merely thought of as the protein responsible for the overall physical nature of the human immunodeficiency virus type 1 (HIV-1), the Gag polyprotein has since been elucidated to have several roles in viral replication and functionality. Over the years, extensive research into the polyproteins’ structure has revealed that Gag can mediate its own trafficking to the plasma membrane, it can interact with several host factors and can even aid in viral genome packaging. Not surprisingly, Gag has also been associated with HIV-1 drug resistance and even treatment failure. Therefore, this review provides an extensive overview of the structural and functional roles of the HIV-1 Gag domains in virion integrity, functionality and infectivity.

Preservation of HIV-1 Gag Helical Bundle Symmetry by Bevirimat Is Central to Maturation Inhibition.

The assembly and maturation of human immunodeficiency virus type 1 (HIV-1) require proteolytic cleavage of the Gag polyprotein. The rate-limiting step resides at the junction between the capsid protein CA and spacer peptide 1, which assembles as a six-helix bundle (6HB). Bevirimat (BVM), the first-in-class maturation inhibitor drug, targets the 6HB and impedes proteolytic cleavage, yet the molecular mechanisms of its activity, and relatedly, the escape mechanisms of mutant viruses, remain unclear. Here, we employed extensive molecular dynamics (MD) simulations and free energy calculations to quantitatively investigate molecular structure-activity relationships, comparing wild-type and mutant viruses in the presence and absence of BVM and inositol hexakisphosphate (IP6), an assembly cofactor. Our analysis shows that the efficacy of BVM is directly correlated with preservation of 6-fold symmetry in the 6HB, which exists as an ensemble of structural states. We identified two primary escape mechanisms, and both lead to loss of symmetry, thereby facilitating helix uncoiling to aid access of protease. Our findings also highlight specific interactions that can be targeted for improved inhibitor activity and support the use of MD simulations for future inhibitor design.

Residues T48 and A49 in HIV-1 NL4-3 Nef are responsible for the counteraction of autophagy initiation, which prevents the ubiquitin-dependent degradation of Gag through autophagosomes

BackgroundAutophagy plays an important role as a cellular defense mechanism against intracellular pathogens, like viruses. Specifically, autophagy orchestrates the recruitment of specialized cargo, including viral components needed for replication, for lysosomal degradation. In addition to this primary role, the cleavage of viral structures facilitates their association with pattern recognition receptors and MHC-I/II complexes, which assists in the modulation of innate and adaptive immune responses against these pathogens. Importantly, whereas autophagy restricts the replicative capacity of human immunodeficiency virus type 1 (HIV-1), this virus has evolved the gene nef to circumvent this process through the inhibition of early and late stages of the autophagy cascade. Despite recent advances, many details of the mutual antagonism between HIV-1 and autophagy still remain unknown. Here, we uncover the genetic determinants that drive the autophagy-mediated restriction of HIV-1 as well as the counteraction imposed by Nef. Additionally, we also examine the implications of autophagy antagonism in HIV-1 infectivity.ResultsWe found that sustained activation of autophagy potently inhibits HIV-1 replication through the degradation of HIV-1 Gag, and that this effect is more prominent for nef-deficient viruses. Gag re-localizes to autophagosomes where it interacts with the autophagosome markers LC3 and SQSTM1. Importantly, autophagy-mediated recognition and recruitment of Gag requires the myristoylation and ubiquitination of this virus protein, two post-translational modifications that are essential for Gag’s central role in virion assembly and budding. We also identified residues T48 and A49 in HIV-1 NL4-3 Nef as responsible for impairing the early stages of autophagy. Finally, a survey of pandemic HIV-1 transmitted/founder viruses revealed that these isolates are highly resistant to autophagy restriction.ConclusionsThis study provides evidence that autophagy antagonism is important for virus replication and suggests that the ability of Nef to counteract autophagy may have played an important role in mucosal transmission. Hence, disabling Nef in combination with the pharmacological manipulation of autophagy represents a promising strategy to prevent HIV spread.

Interferon Induction By HbS, SERINC5 Upregulation and Reduction of HIV-1 Nef and AP2 Contribute to HIV-1 Restriction in Sickle Cell Disease

Concerted action of numerous positively acting cellular factors is essential for Human immunodeficiency virus type 1 (HIV-1) replication but in turn is challenged by anti-viral restriction factors. Previously we showed that ex vivo one round HIV-1 replication and replication of fully competent T-tropic HIV-1(IIIB) is significantly reduced in peripheral blood mononuclear cells (PBMCs) obtained from patients with Sickle Cell Disease (SCD). Further, we identified and confirmed CDKN1A (p21) and CH25H as host restriction factors expressed in SCD PBMCs that may contribute to the HIV-1 inhibition, in addition to the previously reported SAMHD1 and IKBα. Since CH25H is an interferon stimulated gene (ISG), we analyzed IRFs and interferon expression in SCD PBMCs. Higher levels of IRF7 and IFNβ mRNA were observed in SCD PBMCs compared to controls. We probed further to ascertain if hemin or sickle Hb was responsible for interferon response. We found upregulation of IFNβ in THP-1 - derived macrophages treated with lysates of HbSS RBCs or purified HbS as compared to untreated or HbA treated controls. HbSS RBCs lysates and purified HbS inhibited HIV-1 gag mRNA expression in monocyte-derived macrophages infected with HIV-1(Ba-L). Recent clinical study showed increased levels of CD4 in HIV-1 infected SCD patients in Africa. Thus we analyzed CD4 levels in HIV-1 IIIB infected SCD PBMCs, and found them to be higher compared to controls. Levels of HIV-1 nef mRNA, that controls CD4 expression was lower in HIV-1 IIIB infected SCD PBMCs. As Nef counteracts SERINC3/5 restriction factor, we analyzed its expression as well as the expression of AP2 clathrin adaptor that is required for Nef mediated internalization of CD4. AP2 expression was lower and SERINC5 expression was higher in SCD PBMCs. CONCLUSIONS: SCD PBMCs could resist HIV-1 infection because of the increased IFNβ production by macrophages exposed to HbSS or sickle cell RBCs. SCD PBMC have increased levels of SERNIC5 and lower levels of HIV-1 Nef and host AP2 expression that, culumlatively, can increased CD4 levels and lead to the overall improved immunological health of SCD patients. ACKNOWLEDGMENTS: This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005, 1SC1HL150685, 5U54MD007597, 1UM1AI26617 and P30AI087714). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosures No relevant conflicts of interest to declare.

CBP80/20-dependent translation initiation factor (CTIF) inhibits HIV-1 Gag synthesis by targeting the function of the viral protein Rev

ABSTRACT Translation initiation of the human immunodeficiency virus type-1 (HIV-1) full-length RNA has been shown to occur through cap-dependent and IRES-driven mechanisms. Previous studies suggested that the nuclear cap-binding complex (CBC) rather than eIF4E drives cap-dependent translation of the full-length RNA and we have recently reported that the CBC subunit CBP80 supports the function of the viral protein Rev during nuclear export and translation of this viral transcript. Ribosome recruitment during CBC-dependent translation of cellular mRNAs relies on the activity CBP80/20 translation initiation factor (CTIF), which bridges CBP80 and the 40S ribosomal subunit through interactions with eIF3g. Here, we report that CTIF inhibits HIV-1 and HIV-2 Gag synthesis from the full-length RNA. Our results indicate that CTIF associates with HIV-1 Rev through its N-terminal domain and is recruited onto the full-length RNA ribonucleoprotein complex in order to interfere with Gag synthesis. We also demonstrate that CTIF induces the cytoplasmic accumulation of Rev impeding the association of the viral protein with CBP80. We finally show that Rev interferes with the association of CTIF with CBP80 indicating that CTIF and Rev compete for the CBC subunit.

Comparison of HIV-1 Gag and NCp7 in their selectivity for package signal, affinity for stem-loop 3, and Zn2+ content

The human immunodeficiency virus type 1 (HIV-1) Gag recognizes viral packaging signal (Psi) specifically via its nucleocapsid (NC) domain, resulting in the encapsidation of two copies of genomic RNA (gRNA) into the viral particle. The NCp7, which is cleaved from Gag during viral maturation, is a nucleic acid chaperone, coating and protecting the gRNA. In this study, an RT-qPCR-based approach was developed to quantitatively compare the Psi-selectivity of Gag and NCp7 in the presence of bacterial or 293T total RNAs. The binding affinity of Gag and NCp7 to the stem-loop (SL) 3 of Psi was also compared using surface plasmon resonance. We found that Gag selected more Psi-RNA than NCp7 from both E. coli BL21 (DE3) and in vitro binding reactions, and Gag bound to SL3-RNA with a higher affinity than NCp7. Moreover, Gag contained two Zn2+ whereas NCp7 contained one. The N-terminal zinc-finger motif of NCp7 lost most of its Zn2+-binding activity. Deletion of N-terminal amino acids 1–11 of NCp7 resulted in increased Psi-selectivity, SL3-affinity and Zn2+ content. These results indicated that Zn2+ coordination of Gag is critical for Psi-binding and selection. Removal of Zn2+ from the first zinc-finger motif during or after Gag cleavage to generate mature NCp7 might serve as a switch to regulate the functions of Gag NC domain and mature NCp7. Our study will be helpful to elucidate the important roles that Zn2+ plays in the viral life cycle, and may benefit further investigations of the function of HIV-1 Gag and NCp7.

Toward a Macaque Model of HIV-1 Infection: Roadblocks, Progress, and Future Strategies.

The human-specific tropism of Human Immunodeficiency Virus Type 1 (HIV-1) has complicated the development of a macaque model of HIV-1 infection/AIDS that is suitable for preclinical evaluation of vaccines and novel treatment strategies. Several innate retroviral restriction factors, such as APOBEC3 family of proteins, TRIM5α, BST2, and SAMHD1, that prevent HIV-1 replication have been identified in macaque cells. Accessory proteins expressed by Simian Immunodeficiency virus (SIV) such as viral infectivity factor (Vif), viral protein X (Vpx), viral protein R (Vpr), and negative factor (Nef) have been shown to play key roles in overcoming these restriction factors in macaque cells. Thus, substituting HIV-1 accessory genes with those from SIV may enable HIV-1 replication in macaques. We and others have constructed macaque-tropic HIV-1 derivatives [also called simian-tropic HIV-1 (stHIV-1) or Human-Simian Immunodeficiency Virus (HSIV)] carrying SIV vif to overcome APOBEC3 family proteins. Additional modifications to HIV-1 gag in some of the macaque-tropic HIV-1 have also been done to overcome TRIM5α restriction in rhesus and cynomolgus macaques. Although these viruses replicate persistently in macaque species, they do not result in CD4 depletion. Thus, these studies suggest that additional blocks to HIV-1 replication exist in macaques that prevent high-level viral replication. Furthermore, serial animal-to-animal passaging of macaque-tropic HIV-1 in vivo has not resulted in pathogenic variants that cause AIDS in immunocompetent macaques. In this review, we discuss recent developments made toward developing macaque model of HIV-1 infection.

Production of Vesicular Stomatitis Virus Glycoprotein-Pseudotyped Lentiviral Vector Is Enhanced by Ezrin Silencing.

Human immunodeficiency virus type 1 (HIV-1)-based viral vector is widely used as a biomaterial to transfer a gene of interest into target cells in many biological study fields including gene therapy. Vesicular stomatitis virus glycoprotein (VSV-G)-containing HIV-1 vector much more efficiently transduces various mammalian cells than other viral envelope proteins-containing vectors. Understanding the mechanism would contribute to development of a novel method of efficient HIV-1 vector production. HIV-1 vector is generally constructed by transient transfection of human 293T or African green monkey COS7 cells. It was found in this study that HIV-1 Gag protein is constitutively digested in lysosomes of African green monkey cells. Surprisingly, VSV-G elevated HIV-1 Gag protein levels, suggesting that VSV-G protects Gag protein from the lysosomal degradation. Unphosphorylated ezrin, but not phosphorylated ezrin, was detected in COS7 cells, and ezrin silencing elevated Gag protein levels in the presence of VSV-G. Expression of unphosphorylated ezrin reduced Gag protein amounts. These results indicate that unphosphorylated ezrin proteins inhibit the VSV-G-mediated stabilization of HIV-1 Gag protein. Trafficking of HIV-1 Gag-associated intracellular vesicles may be controlled by ezrin. Finally, this study found that ezrin silencing yields higher amount of VSV-G-pseudotyped HIV-1 vector.

The Spirocyclic Imine from a Marine Benthic Dinoflagellate, Portimine, Is a Potent Anti-Human Immunodeficiency Virus Type 1 Therapeutic Lead Compound.

In this study, we aimed to find chemicals from lower sea animals with defensive effects against human immunodeficiency virus type 1 (HIV-1). A library of marine natural products consisting of 80 compounds was screened for activity against HIV-1 infection using a luciferase-encoding HIV-1 vector. We identified five compounds that decreased luciferase activity in the vector-inoculated cells. In particular, portimine, isolated from the benthic dinoflagellate Vulcanodinium rugosum, exhibited significant anti-HIV-1 activity. Portimine inhibited viral infection with an 50% inhibitory concentration (IC50) value of 4.1 nM and had no cytotoxic effect on the host cells at concentrations less than 200 nM. Portimine also inhibited vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped HIV-1 vector infection. This result suggested that portimine mainly targeted HIV-1 Gag or Pol protein. To analyse which replication steps portimine affects, luciferase sequences were amplified by semi-quantitative PCR in total DNA. This analysis revealed that portimine inhibits HIV-1 vector infection before or at the reverse transcription step. Portimine has also been shown to have a direct effect on reverse transcriptase using an in vitro reverse transcriptase assay. Portimine efficiently inhibited HIV-1 replication and is a potent lead compound for developing novel therapeutic drugs against HIV-1-induced diseases.

Restriction of HIV-1 Infection in Sickle Cell Disease and Trait

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) replication is controlled by host intrinsic antiviral restriction factors some of which are counteracted by HIV-1 accessory proteins. We recently showed that ex vivo HIV-1 infection is suppressed in PBMCs obtained from Sickle Cell Disease (SCD) patients. The inhibition was mediated in part by SAMHD1 and NF-κB inhibitor, IkBα and triggered by ferroportin, an iron export protein. Ferroportin expression reduced intracellular iron levels and inhibited cellular CDK2 activity leading to reduction of SAMHD1 phosphorylation and increased expression of IkBα.OBJECTIVES: The study was designed to further clarify the mechanism of HIV-1 inhibition in SCD and identify additional HIV-1 restriction factors that may contribute to the restriction mechanism.METHODS: A customized array was utilized to determine the expression of restriction factors in SCD PBMCs. The shRNA-mediated knockdowns of the identified genes were used to further validate the role of these factors in HIV-1 replication in cultured and primary cells. HIV-1(IIIB strain) and VSVG-pseudotyped pNL4-3.Luc.R-E-virus was used to analyze HIV-1 replication.RESULTS: Overexpression of ferroportin in THP-1 cells led to increased expression of HO-1 and p21 and reduced phosphorylation of SAMHD1. Inhibition of HO-1 and p21 by small molecules increased HIV-1 replication in SCD PBMCs suggesting that these factors contributed to the restriction mechanism. Knocking down SAMHD1 in SCD PBMC did not fully restore HIV-1 replication pointing to additional restriction factors. Analysis of HIV-1 restriction factors in SCD PBMCs using customized array showed increased expression of APOBECs, TRIMs, CH25H, CPSF6, CTR9, EIF2ak2, IFI16, MX2, PML and RTF1 mRNAs. We next tested the effect of SCD trait on HIV-1 infection ex vivo and in vivo. PBMCs obtained from SCD trait individuals showed restricted HIV-1 infection with HIV-1(IIIB strain). To further validate these findings, we compared 9 trait (HbAS) HIV-1 infected individuals with 107 non-SCD (HbAA or HbAC) HIV-1 infected individuals from Howard University HIV clinic. HIV-1 viral load and levels of HIV-1 env and gag were significantly lower in HIV-1 infected SCD trait subjects. Expression of HO-1, p21 were increased and RNR2 expression was reduced in SCD trait PBMCs. Small molecule inhibitors of HO-1 and p21 induced HIV-1 replication in SCD trait PBMCs.CONCLUSIONS: Our findings point to HO-1 and p21 as factors that, in addition to SAMHD1 and IKBα, mediate HIV-1 restriction in SCD. In SCD trait, HO-1, p21 and RNR2 play a key role in ex vivo HIV-1 restriction. Thus HIV-1 infection is deregulated not only in SCD patients but also in SCD trait individuals. The HIV-1 restriction is mediated by iron-activated antiviral restriction factors.ACKNOWLEDGMENTS: This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005, 5G12MD007597, 1UM1AI26617 and P30AI087714). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. DisclosuresNekhai:NIMHD, NIH: Research Funding; NHLBI, NIH: Research Funding; NIAID, NIH: Research Funding.

Single molecule localisation microscopy reveals how HIV-1 Gag proteins sense membrane virus assembly sites in living host CD4 T cells

Monitoring virus assembly at the nanoscale in host cells remains a major challenge. Human immunodeficiency virus type 1 (HIV-1) components are addressed to the plasma membrane where they assemble to form spherical particles of 100 nm in diameter. Interestingly, HIV-1 Gag protein expression alone is sufficient to produce virus-like particles (VLPs) that resemble the immature virus. Here, we monitored VLP formation at the plasma membrane of host CD4+ T cells using a newly developed workflow allowing the analysis of long duration recordings of single-molecule Gag protein localisation and movement. Comparison of Gag assembling platforms in CD4+ T cells expressing wild type or assembly-defective Gag mutant proteins showed that VLP formation lasts roughly 15 minutes with an assembly time of 5 minutes. Trapping energy maps, built from membrane associated Gag protein movements, showed that one third of the assembling energy is due to direct Gag capsid-capsid interaction while the remaining two thirds require the nucleocapsid-RNA interactions. Finally, we show that the viral RNA genome does not increase the attraction of Gag at the membrane towards the assembling site but rather acts as a spatiotemporal coordinator of the membrane assembly process.

Viral subtyping of HIV-1 derived from infected, drug-naive individuals in Jakarta, Indonesia

Human immunodeficiency virus (HIV) infection and acquired immune deficiency syndrome (AIDS) (HIV/AIDS) remain a serious health problem in Indonesia. Jakarta has the largest number of individuals infected with HIV type 1 (HIV-1) in Indonesia; however, data on viral subtyping of HIV-1 is limited. We conducted a molecular epidemiological study on HIV-1 genes derived from infected, drug-naive individuals residing in Jakarta, the capital of Indonesia with large communities of HIV-infected individuals. To determine the distribution of HIV-1 subtypes circulating in Jakarta, 43 HIV-1-infected individuals were recruited in Jakarta, Indonesia. HIV-1 gag and envgenes were amplified from peripheral blood samples derived from study participants. Viral subtyping using the Recombinant Identification Program (RIP) and phylogenetic tree analysis were conducted. RIP and phylogenetic tree analysis revealed that CRF01_AE was the dominant subtype in Jakarta (40%), followed by recombinant viruses containing CRF01_AE and subtype B gene fragments (7%) and subtype B (7%). This study reported the emergence of CRF 01_AE as predominant subtype among HIV-1-infected, drug-naïve individuals in Jakarta, Indonesia. Therefore, continuous surveillance is required in order to detect the emergence of HIV-1-infection.