ATG5 selectively engages virus-tethered BST2/tetherin in an LC3C-associated pathway.

Bone marrow stromal antigen 2 (BST2)/tetherin is a restriction factor that reduces HIV-1 dissemination by tethering virus at the cell surface. BST2 also acts as a sensor of HIV-1 budding, establishing a cellular antiviral state. The HIV-1 Vpu protein antagonizes BST2 antiviral functions via multiple mechanisms, including the subversion of an LC3C-associated pathway, a key cell intrinsic antimicrobial mechanism. Here, we describe the first step of this viral-induced LC3C-associated process. This process is initiated at the plasma membrane through the recognition and internalization of virus-tethered BST2 by ATG5, an autophagy protein. ATG5 and BST2 assemble as a complex, independently of the viral protein Vpu and ahead of the recruitment of the ATG protein LC3C. The conjugation of ATG5 with ATG12 is dispensable for this interaction. ATG5 recognizes cysteine-linked homodimerized BST2 and specifically engages phosphorylated BST2 tethering viruses at the plasma membrane, in an LC3C-associated pathway. We also found that this LC3C-associated pathway is used by Vpu to attenuate the inflammatory responses mediated by virion retention. Overall, we highlight that by targeting BST2 tethering viruses, ATG5 acts as a signaling scaffold to trigger an LC3C-associated pathway induced by HIV-1 infection.

Human retroviral antisense mRNAs are retained in the nuclei of infected cells for viral persistence.

Human retroviruses, including human T cell leukemia virus type 1 (HTLV-1) and HIV type 1 (HIV-1), encode an antisense gene in the negative strand of the provirus. Besides coding for proteins, the messenger RNAs (mRNAs) of retroviral antisense genes have also been found to regulate transcription directly. Thus, it has been proposed that retroviruses likely localize their antisense mRNAs to the nucleus in order to regulate nuclear events; however, this opposes the coding function of retroviral antisense mRNAs that requires a cytoplasmic localization for protein translation. Here, we provide direct evidence that retroviral antisense mRNAs are localized predominantly in the nuclei of infected cells. The retroviral 3' LTR induces inefficient polyadenylation and nuclear retention of antisense mRNA. We further reveal that retroviral antisense RNAs retained in the nucleus associate with chromatin and have transcriptional regulatory function. While HTLV-1 antisense mRNA is recruited to the promoter of C-C chemokine receptor type 4 (CCR4) and enhances transcription from it to support the proliferation of HTLV-1-infected cells, HIV-1 antisense mRNA is recruited to the viral LTR and inhibits sense mRNA expression to maintain the latency of HIV-1 infection. In summary, retroviral antisense mRNAs are retained in nucleus, act like long noncoding RNAs instead of mRNAs, and contribute to viral persistence.

Detection of the HIV-1 Accessory Proteins Nef and Vpu by Flow Cytometry Represents a New Tool to Study Their Functional Interplay within a Single Infected CD4+ T Cell.

The HIV-1 Nef and Vpu accessory proteins are known to protect infected cells from antibody-dependent cellular cytotoxicity (ADCC) responses by limiting exposure of CD4-induced (CD4i) envelope (Env) epitopes at the cell surface. Although both proteins target the host receptor CD4 for degradation, the extent of their functional redundancy is unknown. Here, we developed an intracellular staining technique that permits the intracellular detection of both Nef and Vpu in primary CD4+ T cells by flow cytometry. Using this method, we show that the combined expression of Nef and Vpu predicts the susceptibility of HIV-1-infected primary CD4+ T cells to ADCC by HIV+ plasma. We also show that Vpu cannot compensate for the absence of Nef, thus providing an explanation for why some infectious molecular clones that carry a LucR reporter gene upstream of Nef render infected cells more susceptible to ADCC responses. Our method thus represents a new tool to dissect the biological activity of Nef and Vpu in the context of other host and viral proteins within single infected CD4+ T cells. IMPORTANCE HIV-1 Nef and Vpu exert several biological functions that are important for viral immune evasion, release, and replication. Here, we developed a new method allowing simultaneous detection of these accessory proteins in their native form together with some of their cellular substrates. This allowed us to show that Vpu cannot compensate for the lack of a functional Nef, which has implications for studies that use Nef-defective viruses to study ADCC responses.

A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor.

The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu's itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 was supported by STAM and PTPN23 and to a much lesser extent by the retromer subunits Vps35 and SNX3. PTPN23 also supported the Vpu-directed decrease in CD4 at the cell surface. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.

Factors that mold the nuclear landscape of HIV-1 integration.

The integration of retroviral reverse transcripts into the chromatin of the cells that they infect is required for virus replication. Retroviral integration has far-reaching consequences, from perpetuating deadly human diseases to molding metazoan evolution. The lentivirus human immunodeficiency virus 1 (HIV-1), which is the causative agent of the AIDS pandemic, efficiently infects interphase cells due to the active nuclear import of its preintegration complex (PIC). To enable integration, the PIC must navigate the densely-packed nuclear environment where the genome is organized into different chromatin states of varying accessibility in accordance with cellular needs. The HIV-1 capsid protein interacts with specific host factors to facilitate PIC nuclear import, while additional interactions of viral integrase, the enzyme responsible for viral DNA integration, with cellular nuclear proteins and nucleobases guide integration to specific chromosomal sites. HIV-1 integration favors transcriptionally active chromatin such as speckle-associated domains and disfavors heterochromatin including lamina-associated domains. In this review, we describe virus-host interactions that facilitate HIV-1 PIC nuclear import and integration site targeting, highlighting commonalities among factors that participate in both of these steps. We moreover discuss how the nuclear landscape influences HIV-1 integration site selection as well as the establishment of active versus latent virus infection.

The viral protein U (Vpu)-interacting host protein ATP6V0C down-regulates cell-surface expression of tetherin and thereby contributes to HIV-1 release.

Host proteins with antiviral activity have evolved as first-line defenses to suppress viral replication. The HIV-1 accessory protein viral protein U (Vpu) enhances release of the virus from host cells by down-regulating the cell-surface expression of the host restriction factor tetherin. However, the exact mechanism of Vpu-mediated suppression of antiviral host responses is unclear. To further understand the role of host proteins in Vpu's function, here we carried out yeast two-hybrid screening and identified the V0 subunit C of vacuolar ATPase (ATP6V0C) as a Vpu-binding protein. To examine the role of ATP6V0C in Vpu-mediated tetherin degradation and HIV-1 release, we knocked down ATP6V0C expression in HeLa cells and observed that ATP6V0C depletion impairs Vpu-mediated tetherin degradation, resulting in defective HIV-1 release. We also observed that ATP6V0C overexpression stabilizes tetherin expression. This stabilization effect was specific to ATP6V0C, as overexpression of another subunit of the vacuolar ATPase, ATP6V0C″, had no effect on tetherin expression. ATP6V0C overexpression did not stabilize CD4, another target of Vpu-mediated degradation. Immunofluorescence localization experiments revealed that the ATP6V0C-stabilized tetherin is sequestered in a CD63- and lysosome-associated membrane protein 1 (LAMP1)-positive intracellular compartment. These results indicate that the Vpu-interacting protein ATP6V0C plays a role in down-regulating cell-surface expression of tetherin and thereby contributes to HIV-1 assembly and release.

Differential Vpu-Mediated CD4 and Tetherin Downregulation Functions among Major HIV-1 Group M Subtypes.

Downregulation of BST-2/tetherin and CD4 by HIV-1 viral protein U (Vpu) promotes viral egress and allows infected cells to evade host immunity. Little is known however about the natural variability in these Vpu functions among the genetically diverse viral subtypes that contribute to the HIV-1 pandemic. We collected Vpu isolates from 332 treatment-naive individuals living with chronic HIV-1 infection in Uganda, Rwanda, South Africa, and Canada. Together, these Vpu isolates represent four major HIV-1 group M subtypes (A [n = 63], B [n = 84], C [n = 94], and D [n = 59]) plus intersubtype recombinants and uncommon strains (n = 32). The ability of each Vpu clone to downregulate endogenous CD4 and tetherin was quantified using flow cytometry following transfection into an immortalized T-cell line and compared to that of a reference Vpu clone derived from HIV-1 subtype B NL4.3. Overall, the median CD4 downregulation function of natural Vpu isolates was similar to that of NL4.3 (1.01 [interquartile range {IQR}, 0.86 to 1.18]), while the median tetherin downregulation function was moderately lower than that of NL4.3 (0.90 [0.79 to 0.97]). Both Vpu functions varied significantly among HIV-1 subtypes (Kruskal-Wallis P < 0.0001). Specifically, subtype C clones exhibited the lowest CD4 and tetherin downregulation activities, while subtype D and B clones were most functional for both activities. We also identified Vpu polymorphisms associated with CD4 or tetherin downregulation function and validated six of these using site-directed mutagenesis. Our results highlight the marked extent to which Vpu function varies among global HIV-1 strains, raising the possibility that natural variation in this accessory protein may contribute to viral pathogenesis and/or spread.IMPORTANCE The HIV-1 accessory protein Vpu enhances viral spread by downregulating CD4 and BST-2/tetherin on the surface of infected cells. Natural variability in these Vpu functions may contribute to HIV-1 pathogenesis, but this has not been investigated among the diverse viral subtypes that contribute to the HIV-1 pandemic. In this study, we found that Vpu function differs significantly among HIV-1 subtypes A, B, C, and D. On average, subtype C clones displayed the lowest ability to downregulate both CD4 and tetherin, while subtype B and D clones were more functional. We also identified Vpu polymorphisms that associate with functional differences among HIV-1 isolates and subtypes. Our study suggests that genetic diversity in Vpu may play an important role in the differential pathogenesis and/or spread of HIV-1.

Vpu of a Simian Immunodeficiency Virus Isolated from Greater Spot-Nosed Monkey Antagonizes Human BST-2 via Two AxxxxxxxW Motifs.

BST-2/CD317/tetherin is a host transmembrane protein that potently inhibits human immunodeficiency virus type 1 (HIV-1) virion release by tethering the nascent virions to the plasma membrane. Viral protein U (Vpu) is an accessory protein encoded by HIV-1 as well as by some simian immunodeficiency viruses (SIVs) infecting wild chimpanzees, gorillas, or monkeys (SIVcpz, SIVgor, or SIVgsn/SIVmon/SIVmus, respectively). HIV-1 Vpu directly binds to and downregulates human BST-2. The antagonism is highly species specific because the amino acid sequences of BST-2 are different among animal species. Here, we show that Vpu proteins from several SIVcpz, SIVgsn, SIVmon, or SIVmus isolates fail to antagonize human BST-2. Only Vpu from an SIVgsn isolate (SIVgsn-99CM71 [SIVgsn71]) was able to antagonize human BST-2 as well as BST-2 of its natural host, greater spot-nosed monkey (GSN). This SIVgsn Vpu interacted with human BST-2, downregulated cell surface human BST-2 expression, and facilitated HIV-1 virion release in the presence of human BST-2. While the unique 14AxxxxxxxW22 motif in the transmembrane domain of HIV-1NL4-3Vpu was reported to be important for antagonizing human BST-2, we show here that two AxxxxxxxW motifs (A22W30 and A25W33) exist in SIVgsn71 Vpu. Only the A22W30 motif was needed for SIVgsn71 Vpu to antagonize GSN BST-2, suggesting that the mechanism of this antagonism resembles that of HIV-1NL4-3 Vpu against human BST-2. Interestingly, SIVgsn71 Vpu requires two AxxxxxxxW (A22W30 and A25W33) motifs to antagonize human BST-2, suggesting an as-yet-undefined way that SIVgsn71 Vpu works against human BST-2. These results imply an evolutionary impact of primate BST-2 on lentiviral Vpu.IMPORTANCE Genetic alterations conferring a selective advantage in protecting from life-threating pathogens are maintained during evolution. In fact, the amino acid sequences of BST-2 differ among primate animals and their susceptibility to viral proteins is species specific, suggesting that such genetic diversity has arisen through the evolutionarily controlled balance between the host and pathogens. The M (main) group of HIV-1 is thought to be derived from SIVcpz, which utilizes Nef, but not Vpu, to antagonize chimpanzee BST-2. SIVcpz Nef is, however, unable to antagonize human BST-2, and Vpu was consequently chosen again as an antagonist against human BST-2 in the context of HIV-1. Studies on how Vpu lost and acquired this ability, together with the distinct mechanisms by which SIVgsn71 Vpu binds to and downregulates human or GSN BST-2, may help to explain the evolution of this lentiviral protein as a result of host-pathogen interactions.

Recombinant expression, purification, and characterization of full-length human BST-2 from Escherichia coli.

HIV-1 virus release from infected cells is blocked by human BST-2, but HIV-1 Vpu efficiently antagonises BST-2 due to direct transmembrane domain interactions that occur between each protein. Targeting the interaction between these two proteins is seen as viable for HIV-1 antiviral intervention. This study describes the successful over-expression and purification of a recombinant full-length human BST-2 from inclusion bodies using affinity and anion exchange chromatography. Two milligrams of purified full-length BST-2 were produced per litre of BL21 (DE3) T7 Express® pLysY E. coli culture. Far-UV circular dichroism validated the renaturing of the recombinant protein and retention of its secondary structure. Furthermore, through ELISA, a known human BST-2 binding partner, HIV-1 Vpu, was shown to bind to the renatured and purified protein, further validating its folding. To our knowledge this is the first report of the purification of a wild-type, full-length human BST-2 from Escherichia coli.

Fast hierarchical Bayesian analysis of population structure.

We present fastbaps, a fast solution to the genetic clustering problem. Fastbaps rapidly identifies an approximate fit to a Dirichlet process mixture model (DPM) for clustering multilocus genotype data. Our efficient model-based clustering approach is able to cluster datasets 10-100 times larger than the existing model-based methods, which we demonstrate by analyzing an alignment of over 110 000 sequences of HIV-1 pol genes. We also provide a method for rapidly partitioning an existing hierarchy in order to maximize the DPM model marginal likelihood, allowing us to split phylogenetic trees into clades and subclades using a population genomic model. Extensive tests on simulated data as well as a diverse set of real bacterial and viral datasets show that fastbaps provides comparable or improved solutions to previous model-based methods, while being significantly faster. The method is made freely available under an open source MIT licence as an easy to use R package at https://github.com/gtonkinhill/fastbaps.

In silico design and in vitro expression of novel multiepitope DNA constructs based on HIV-1 proteins and Hsp70 T-cell epitopes.

OBJECTIVES: Epitope-driven vaccines carrying highly conserved and immunodominant epitopes have emerged as promising approaches to overcome human immunodeficiency virus-1 (HIV-1) infection. METHODS: Two multiepitope DNA constructs encoding T cell epitopes from HIV-1 Gag, Pol, Env, Nef and Rev proteins alone and/or linked to the immunogenic epitopes derived from heat shock protein 70 (Hsp70) as an immunostimulatory agent were designed. In silico analyses were applied including MHC-I and MHC-II binding, MHC-I immunogenicity and antigen processing, population coverage, conservancy, allergenicity, toxicity and hemotoxicity. The peptide-MHC-I/MHC-II molecular docking and cytokine production analyses were carried out for predicted epitopes. The selected highly immunogenic T-cell epitopes were then used to design two multiepitope fusion constructs. Next, prediction of the physicochemical and structural properties, B cell epitopes, and constructs-toll-like receptors (TLRs) molecular docking were performed for each construct. Finally, the eukaryotic expression plasmids harboring totally 12 cytotoxic T Lymphocyte (CTL) and 10 helper T lymphocytes (HTL) epitopes from HIV-1 proteins (i.e., pEGFP-N1-gag-pol-env-nef-rev), and linked to 2 CTL and 2 HTL epitopes from Hsp70 (i.e., pEGFP-N1-hsp70-gag-pol-env-nef-rev) were generated and transfected into HEK-293 T cells for evaluating the percentage of multiepitope peptides expression using flow cytometry and western blotting. RESULTS: The designed DNA constructs could be successfully expressed in mammalian cells. The expression rates of Gag-Pol-Env-Nef-Rev-GFP and Hsp70-Gag-Pol-Env-Nef-Rev-GFP were about 56-60% as the bands of ~ 63 and ~ 72 kDa confirmed in western blotting, respectively. CONCLUSION: The combined in silico/in vitro methods indicated two multiepitope constructs can be produced and used as probable effective immunogens for HIV-1 vaccine development.

A technique for delineating the unfolding requirements for substrate entry into retrotranslocons during endoplasmic reticulum-associated degradation.

The endoplasmic reticulum-associated degradation (ERAD) pathway mediates the endoplasmic reticulum-to-cytosol retrotranslocation of defective proteins through protein complexes called retrotranslocons. Defective proteins usually have complex conformations and topologies, and it is unclear how ERAD can thread these conformationally diverse protein substrates through the retrotranslocons. Here, we investigated the substrate conformation flexibility necessary for transport via retrotranslocons on the ERAD-L, ERAD-M, and HIV-encoded protein Vpu-hijacked ERAD branches. To this end, we appended various ERAD substrates with specific domains whose conformations were tunable in flexibility or tightness by binding to appropriate ligands. With this technique, we could define the capacity of specific retrotranslocons in disentangling very tight, less tight but well-folded, and unstructured conformations. The Hrd1 complex, the retrotranslocon on the ERAD-L branch, permitted the passage of substrates with a proteinase K-resistant tight conformation, whereas the E3 ligase gp78-mediated ERAD-M allowed passage only of nearly completely disordered but not well-folded substrates and thus may have the least unfoldase activity. Vpu-mediated ERAD, containing a potential retrotranslocon, could unfold well-folded substrates for successful retrotranslocation. However, substrate retrotranslocation in Vpu-mediated ERAD was blocked by enhanced conformational tightness of the substrate. On the basis of these findings, we propose a mechanism underlying polypeptide movement through the endoplasmic reticulum membrane. We anticipate that our biochemical system paves the way for identifying the factors necessary for the retrotranslocation of membrane proteins.

The C-Terminal End of HIV-1 Vpu Has a Clade-Specific Determinant That Antagonizes BST-2 and Facilitates Virion Release.

The cellular protein bone marrow stromal antigen-2 (BST-2)/tetherin acts against a variety of enveloped viruses by restricting their release from the plasma membrane. The HIV-1 accessory protein Vpu counteracts BST-2 by downregulating it from the cell surface and displacing it from virion assembly sites. Previous comparisons of Vpus from transmitted/founder viruses and between viruses isolated during acute and chronic infection led to the identification of a tryptophan at position 76 in Vpu (W76) as a key determinant for the displacement of BST-2 from virion assembly sites. Although present in Vpus from clades B, D, and G, W76 is absent from Vpus from clades A, C, and H. Mutagenesis of the C-terminal region of Vpu from two clade C viruses led to the identification of a conserved LL sequence that is functionally analogous to W76 of clade B. Alanine substitution of these leucines partially impaired virion release. This impairment was even greater when the mutations were combined with mutations of the Vpu ß-TrCP binding site, resulting in Vpu proteins that induced high surface levels of BST-2 and reduced the efficiency of virion release to less than that of virus lacking vpu Microscopy confirmed that these C-terminal leucines in clade C Vpu, like W76 in clade B, contribute to virion release by supporting the displacement of BST-2 from virion assembly sites. These results suggest that although encoded differently, the ability of Vpu to displace BST-2 from sites of virion assembly on the plasma membrane is evolutionarily conserved among clade B and C HIV-1 isolates.IMPORTANCE Although targeted by a variety of restriction mechanisms, HIV-1 establishes chronic infection in most cases, in part due to the counteraction of these host defenses by viral accessory proteins. Using conserved motifs, the accessory proteins exploit the cellular machinery to degrade or mistraffic host restriction factors, thereby counteracting them. The Vpu protein counteracts the virion-tethering factor BST-2 in part by displacing it from virion assembly sites along the plasma membrane, but a previously identified determinant of that activity is clade specific at the level of protein sequence and not found in the clade C viruses that dominate the pandemic. Here, we show that clade C Vpu provides this activity via a leucine-containing sequence rather than the tryptophan-containing sequence found in clade B Vpu. This difference seems likely to reflect the different evolutionary paths taken by clade B and clade C HIV-1 in human populations.

Genotypic and Mechanistic Characterization of Subtype-Specific HIV Adaptation to Host Cellular Immunity.

The extent to which viral genetic context influences HIV adaptation to human leukocyte antigen (HLA) class I-restricted immune pressures remains incompletely understood. The Ugandan HIV epidemic, where major pandemic group M subtypes A1 and D cocirculate in a single host population, provides an opportunity to investigate this question. We characterized plasma HIV RNA gag, pol, and nef sequences, along with host HLA genotypes, in 464 antiretroviral-naive individuals chronically infected with HIV subtype A1 or D. Using phylogenetically informed statistical approaches, we identified HLA-associated polymorphisms and formally compared their strengths of selection between viral subtypes. A substantial number (32%) of HLA-associated polymorphisms identified in subtype A1 and/or D had previously been reported in subtype B, C, and/or circulating recombinant form 01_AE (CRF01_AE), confirming the shared nature of many HLA-driven escape pathways regardless of viral genetic context. Nevertheless, 34% of the identified HLA-associated polymorphisms were significantly differentially selected between subtypes A1 and D. Experimental investigation of select examples of subtype-specific escape revealed distinct underlying mechanisms with important implications for vaccine design: whereas some were attributable to subtype-specific sequence variation that influenced epitope-HLA binding, others were attributable to differential mutational barriers to immune escape. Overall, our results confirm that HIV genetic context is a key modulator of viral adaptation to host cellular immunity and highlight the power of combined bioinformatic and mechanistic studies, paired with knowledge of epitope immunogenicity, to identify appropriate viral regions for inclusion in subtype-specific and universal HIV vaccine strategies.IMPORTANCE The identification of HIV polymorphisms reproducibly selected under pressure by specific HLA alleles and the elucidation of their impact on viral function can help identify immunogenic viral regions where immune escape incurs a fitness cost. However, our knowledge of HLA-driven escape pathways and their functional costs is largely limited to HIV subtype B and, to a lesser extent, subtype C. Our study represents the first characterization of HLA-driven adaptation pathways in HIV subtypes A1 and D, which dominate in East Africa, and the first statistically rigorous characterization of differential HLA-driven escape across viral subtypes. The results support a considerable impact of viral genetic context on HIV adaptation to host HLA, where HIV subtype-specific sequence variation influences both epitope-HLA binding and the fitness costs of escape. Integrated bioinformatic and mechanistic characterization of these and other instances of differential escape could aid rational cytotoxic T-lymphocyte-based vaccine immunogen selection for both subtype-specific and universal HIV vaccines.

HLA-C downregulation by HIV-1 adapts to host HLA genotype.

HIV-1 can downregulate HLA-C on infected cells, using the viral protein Vpu, and the magnitude of this downregulation varies widely between primary HIV-1 variants. The selection pressures that result in viral downregulation of HLA-C in some individuals, but preservation of surface HLA-C in others are not clear. To better understand viral immune evasion targeting HLA-C, we have characterized HLA-C downregulation by a range of primary HIV-1 viruses. 128 replication competent viral isolates from 19 individuals with effective anti-retroviral therapy, show that a substantial minority of individuals harbor latent reservoir virus which strongly downregulates HLA-C. Untreated infections display no change in HLA-C downregulation during the first 6 months of infection, but variation between viral quasispecies can be detected in chronic infection. Vpu molecules cloned from plasma of 195 treatment naïve individuals in chronic infection demonstrate that downregulation of HLA-C adapts to host HLA genotype. HLA-C alleles differ in the pressure they exert for downregulation, and individuals with higher levels of HLA-C expression favor greater viral downregulation of HLA-C. Studies of primary and mutant molecules identify 5 residues in the transmembrane region of Vpu, and 4 residues in the transmembrane domain of HLA-C, which determine interactions between Vpu and HLA. The observed adaptation of Vpu-mediated downregulation to host genotype indicates that HLA-C alleles differ in likelihood of mediating a CTL response that is subverted by viral downregulation, and that preservation of HLA-C expression is favored in the absence of these responses. Finding that latent reservoir viruses can downregulate HLA-C could have implications for HIV-1 cure therapy approaches in some individuals.

Viral genetic variation accounts for a third of variability in HIV-1 set-point viral load in Europe.

HIV-1 set-point viral load-the approximately stable value of viraemia in the first years of chronic infection-is a strong predictor of clinical outcome and is highly variable across infected individuals. To better understand HIV-1 pathogenesis and the evolution of the viral population, we must quantify the heritability of set-point viral load, which is the fraction of variation in this phenotype attributable to viral genetic variation. However, current estimates of heritability vary widely, from 6% to 59%. Here we used a dataset of 2,028 seroconverters infected between 1985 and 2013 from 5 European countries (Belgium, Switzerland, France, the Netherlands and the United Kingdom) and estimated the heritability of set-point viral load at 31% (CI 15%-43%). Specifically, heritability was measured using models of character evolution describing how viral load evolves on the phylogeny of whole-genome viral sequences. In contrast to previous studies, (i) we measured viral loads using standardized assays on a sample collected in a strict time window of 6 to 24 months after infection, from which the viral genome was also sequenced; (ii) we compared 2 models of character evolution, the classical "Brownian motion" model and another model ("Ornstein-Uhlenbeck") that includes stabilising selection on viral load; (iii) we controlled for covariates, including age and sex, which may inflate estimates of heritability; and (iv) we developed a goodness of fit test based on the correlation of viral loads in cherries of the phylogenetic tree, showing that both models of character evolution fit the data well. An overall heritability of 31% (CI 15%-43%) is consistent with other studies based on regression of viral load in donor-recipient pairs. Thus, about a third of variation in HIV-1 virulence is attributable to viral genetic variation.

B1 protein: a novel cell penetrating protein for in vitro and in vivo delivery of HIV-1 multi-epitope DNA constructs.

OBJECTIVES: Enhancement of the potential ability of biomacromolecules to cross cell membranes is a critical step for development of effective therapeutic vaccine especially DNA vaccine against human immunodeficiency virus-1 (HIV-1) infection. The supercharged proteins were known as powerful weapons for delivery of different types of cargoes such as DNA and protein. Hence, we applied B1 protein with + 43 net charges obtained from a single frameshift in the gene encoding enhanced green fluorescent protein (eGFP) for delivery of two multi-epitope DNA constructs (nef-vpu-gp160-p24 and nef-vif-gp160-p24) in vitro and in vivo for the first time. For this purpose, B1 protein was generated in bacterial expression system under native conditions, and used to interact with both DNA constructs. RESULTS: Our data indicated that B1 protein (~ 27 kDa) was able to form a stable nanoparticle (~ 80-110 nm) with both DNA constructs at nitrogen: phosphate (N: P) ratio of 1:1. Moreover, the transfection efficiency of B1 protein for DNA delivery into HEK-293T cell line indicated that the cellular uptake of nef-vif-gp160-p24 DNA/ B1 and nef-vpu-gp160-p24 DNA/ B1 nanoparticles was about 32-35% with lower intensity as compared to TurboFect commercial reagent. On the other hand, immunization of BALB/c mice with different modalities demonstrated that B1 protein could enhance the levels of antibody, IFN-gamma and Granzyme B eliciting potent and strong Th1-directed cellular immunity. CONCLUSION: Generally, our findings showed the potency of B1 protein as a promising gene delivery system to improve an effective therapeutic vaccine against HIV-1 infection.

Human T-cell leukemia virus type 1 Gag domains have distinct RNA-binding specificities with implications for RNA packaging and dimerization.

Human T-cell leukemia virus type 1 (HTLV-1) is the first retrovirus that has conclusively been shown to cause human diseases. In HIV-1, specific interactions between the nucleocapsid (NC) domain of the Gag protein and genomic RNA (gRNA) mediate gRNA dimerization and selective packaging; however, the mechanism for gRNA packaging in HTLV-1, a deltaretrovirus, is unclear. In other deltaretroviruses, the matrix (MA) and NC domains of Gag are both involved in gRNA packaging, but MA binds nucleic acids with higher affinity and has more robust chaperone activity, suggesting that this domain may play a primary role. Here, we show that the MA domain of HTLV-1, but not the NC domain, binds short hairpin RNAs derived from the putative gRNA packaging signal. RNA probing of the HTLV-1 5' leader and cross-linking studies revealed that the primer-binding site and a region within the putative packaging signal form stable hairpins that interact with MA. In addition to a previously identified palindromic dimerization initiation site (DIS), we identified a new DIS in HTLV-1 gRNA and found that both palindromic sequences bind specifically the NC domain. Surprisingly, a mutant partially defective in dimer formation in vitro exhibited a significant increase in RNA packaging into HTLV-1-like particles, suggesting that efficient RNA dimerization may not be strictly required for RNA packaging in HTLV-1. Moreover, the lifecycle of HTLV-1 and other deltaretroviruses may be characterized by NC and MA functions that are distinct from those of the corresponding HIV-1 proteins, but together provide the functions required for viral replication.

Phosphoserine acidic cluster motifs bind distinct basic regions on the µ subunits of clathrin adaptor protein complexes.

Protein trafficking in the endosomal system involves the recognition of specific signals within the cytoplasmic domains (CDs) of transmembrane proteins by clathrin adaptors. One such signal is the phosphoserine acidic cluster (PSAC), the prototype of which is in the endoprotease furin. How PSACs are recognized by clathrin adaptors has been controversial. We reported previously that HIV-1 Vpu, which modulates cellular immunoreceptors, contains a PSAC that binds to the µ subunits of clathrin adaptor protein (AP) complexes. Here, we show that the CD of furin binds the µ subunits of AP-1 and AP-2 in a phosphorylation-dependent manner. Moreover, we identify a potential PSAC in a cytoplasmic loop of the cellular transmembrane Serinc3, an inhibitor of the infectivity of retroviruses. The two serines within the PSAC of Serinc3 are phosphorylated by casein kinase II and mediate interaction with the µ subunits in vitro The sites of these serines vary among mammals in a manner suggesting host-pathogen conflict, yet the Serinc3 PSAC seems dispensable for anti-HIV activity and for counteraction by HIV-1 Nef. The CDs of Vpu and furin and the PSAC-containing loop of Serinc3 each bind the µ subunit of AP-2 (µ2) with similar affinities, but they appear to utilize different basic regions on µ2. The Serinc3 loop requires a region previously reported to bind the acidic plasma membrane lipid phosphatidylinositol 4,5-bisphosphate. These data suggest that the PSACs within different proteins recognize different basic regions on the µ surface, providing the potential to inhibit the activity of viral proteins without necessarily affecting cellular protein trafficking.