Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu.

Viroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can self-assemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins. Here, we use native mass spectrometry in detergent micelles to uncover the patterns of oligomerization of the full-length SARS-CoV-2 envelope (E) protein, poliovirus VP4, and HIV Vpu. Our data suggest that the E protein is a specific dimer, VP4 is exclusively monomeric, and Vpu assembles into a polydisperse mixture of oligomers under these conditions. Overall, these results revealed the diversity in the oligomerization of viroporins, which has implications for the mechanisms of their biological functions as well as their potential as therapeutic targets.

MicroRNA-25/93 induction by Vpu as a mechanism for counteracting MARCH1-restriction on HIV-1 infectivity in macrophages.

IMPORTANCE: In order to efficiently produce infectious viral particles, HIV must counter several restrictions exerted by host cell antiviral proteins. MARCH1 is a member of the MARCH protein family that restricts HIV infection by limiting the incorporation of viral envelope glycoproteins into nascent virions. Here, we identified two regulatory RNAs, microRNAs-25 and -93, induced by the HIV-1 accessory protein Vpu, that downregulate MARCH1 mRNA. We also show that Vpu induces these cellular microRNAs in macrophages by hijacking the cellular ß-catenin pathway. The notion that HIV-1 has evolved a mechanism to counteract MARCH1 restriction on viral infectivity underlines the importance of MARCH1 in the host antiviral response.

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.

The Feline Immunodeficiency Virus Envelope Signal Peptide Is a Tetherin Antagonizing Protein.

Signal peptides are N-terminal peptides, generally less than 30 amino acids in length, that direct translocation of proteins into the endoplasmic reticulum and secretory pathway. The envelope glycoprotein (Env) of the nonprimate lentivirus feline immunodeficiency virus (FIV) contains the longest signal peptide of all eukaryotic, prokaryotic, and viral proteins (175 amino acids), yet the reason is unknown. Tetherin is a dual membrane-anchored host protein that inhibits the release of enveloped viruses from cells. Primate lentiviruses have evolved three antagonists: the small accessory proteins Vpu and Nef, and in the case of HIV-2, Env. Here, we identify the FIV Env signal peptide (Fsp) as the FIV tetherin antagonist. A short deletion in the central portion of Fsp had no effect on viral replication in the absence of tetherin, but severely impaired virion budding in its presence. Fsp is necessary and sufficient, acting as an autonomous accessory protein with the rest of Env dispensable. In contrast to primate lentivirus tetherin antagonists, its mechanism is to stringently block the incorporation of this restriction factor into viral particles rather than by degrading it or downregulating it from the plasma membrane. IMPORTANCE The study of species- and virus-specific differences in restriction factors and their antagonists has been central to deciphering the nature of these key host defenses. FIV is an AIDS-causing lentivirus that has achieved pandemic spread in the domestic cat. We now identify its tetherin antagonist as the signal sequence of the Envelope glycoprotein, thus identifying the fourth lentiviral anti-tetherin protein and the first new lentiviral accessory protein in decades. Fsp is necessary and sufficient and functions by stringently blocking particle incorporation of tetherin, which differs from the degradation or surface downregulation mechanisms used by primate lentiviruses. Fsp also is a novel example of signal peptide dual function, being both a restriction factor antagonist and a mediator of protein translocation into the endoplasmic reticulum.

Insights into the oligomeric structure of the HIV-1 Vpu protein.

The HIV-1-encoded protein Vpu forms an oligomeric ion channel/pore in membranes and interacts with host proteins to support the virus lifecycle. However, Vpu molecular mechanisms are currently not well understood. Here, we report on the Vpu oligomeric organization under membrane and aqueous conditions and provide insights into how the Vpu environment affects the oligomer formation. For these studies, we designed a maltose-binding protein (MBP)-Vpu chimera protein and produced it in E. coli in soluble form. We analyzed this protein using analytical size-exclusion chromatography (SEC), negative staining electron microscopy (nsEM), and electron paramagnetic resonance (EPR) spectroscopy. Surprisingly, we found that MBP-Vpu formed stable oligomers in solution, seemingly driven by Vpu transmembrane domain self-association. A coarse modeling of nsEM data as well as SEC and EPR data suggests that these oligomers most likely are pentamers, similar to what was reported regarding membrane-bound Vpu. We also noticed reduced MBP-Vpu oligomer stability upon reconstitution of the protein in ß-DDM detergent and mixtures of lyso-PC/PG or DHPC/DHPG. In these cases, we observed greater oligomer heterogeneity, with MBP-Vpu oligomeric order generally lower than in solution; however, larger oligomers were also present. Notably, we found that in lyso-PC/PG, above a certain protein concentration, MBP-Vpu assembles into extended structures, which had not been reported for Vpu. Therefore, we captured various Vpu oligomeric forms, which can shed light on Vpu quaternary organization. Our findings could be useful in understanding Vpu organization and function in cellular membranes and could provide information regarding the biophysical properties of single-pass transmembrane proteins.

Involvement of TRPM7 in Alcohol-Induced Damage of the Blood-Brain Barrier in the Presence of HIV Viral Proteins.

Ethanol (EtOH) exerts its effects through various protein targets, including transient receptor potential melastatin 7 (TRPM7) channels, which play an essential role in cellular homeostasis. We demonstrated that TRPM7 is expressed in rat brain microvascular endothelial cells (rBMVECs), the major cellular component of the blood-brain barrier (BBB). Heavy alcohol drinking is often associated with HIV infection, however mechanisms underlying alcohol-induced BBB damage and HIV proteins, are not fully understood. We utilized the HIV-1 transgenic (HIV-1Tg) rat to mimic HIV-1 patients on combination anti-retroviral therapy (cART) and demonstrated TRPM7 expression in rBMVECs wass lower in adolescent HIV-1Tg rats compared to control animals, however control and HIV-1Tg rats expressed similar levels at 9 weeks, indicating persistent presence of HIV-1 proteins delayed TRPM7 expression. Binge exposure to EtOH (binge EtOH) decreased TRPM7 expression in control rBMVECs in a concentration-dependent manner, and abolished TRPM7 expression in HIV-1Tg rats. In human BMVECs (hBMVECs), TRPM7 expression was downregulated after treatment with EtOH, HIV-1 proteins, and in combination. Next, we constructed in vitro BBB models using BMVECs and found TRPM7 antagonists enhanced EtOH-mediated BBB integrity changes. Our study demonstrated alcohol decreased TRPM7 expression, whereby TRPM7 could be involved in the mechanisms underlying BBB alcohol-induced damage in HIV-1 patients on cART.

An Observational Study of Genetic Diversity of HIV-1 vpu in Rapid Progressors in India.

BACKGROUND: The genetic diversity in HIV-1 genes affects viral pathogenesis in HIV-1 positive patients. Accessory genes of HIV-1, including vpu, are reported to play a critical role in HIV pathogenesis and disease progression. Vpu has a crucial role in CD4 degradation and virus release. The sequence heterogeneity in the vpu gene may affect disease progression in patients, therefore, the current study was undertaken to identify the role of vpu in patients defined as rapid progressors. OBJECTIVE: The objective of the study was to identify the viral determinants present on vpu that may be important in disease progression in rapid progressors. METHODS: Blood samples were collected from 13 rapid progressors. DNA was isolated from PBMCs and vpu was amplified using nested PCR. Both strands of the gene were sequenced using an automated DNA Sequencer. The characterization and analysis of vpu was done using various bioinformatics tools. RESULTS: The analysis revealed that all sequences had intact ORF and sequence heterogeneity was present across all sequences and distributed all over the gene. The synonymous substitutions, however, were higher than nonsynonymous substitutions. The phylogenetic tree analysis showed an evolutionary relationship with previously published Indian subtype C sequences. Comparatively, the cytoplasmic tail(77 - 86) showed the highest degree of variability in these sequences as determined by Entropy- one tool. CONCLUSION: The study showed that due to the robust nature of the protein, the biological activity of the protein was intact and sequence heterogeneity may promote disease progression in the study population.

LL-37 antimicrobial peptide and heterologous prime-boost vaccination regimen significantly induce HIV-1 Nef-Vpr antigen- and virion-specific immune responses in mice.

OBJECTIVES: HIV infection still remains a leading cause of morbidity and mortality worldwide. The inability of highly-active antiretroviral therapy in HIV-1 eradication led to development of therapeutic vaccines. Exploiting effective immunogenic constructs and potent delivery systems are important to generate effective therapeutic vaccines, and overcome their poor membrane permeability. Among HIV-1 proteins, the Nef and Vpr proteins can be considered as antigen candidates in vaccine design. METHODS: In this study, the immunogenicity of Nef-Vpr antigen candidate in different regimens along with antimicrobial peptide LL-37 (as a DNA carrier) and Montanide 720 (as an adjuvant) was studied in mice. Moreover, the secretion of cytokines was assessed in virion-exposed mice lymphocytes in vitro. RESULTS: Our data indicated that groups immunized with the homologous protein + Montanide regimen (group 1), and also the heterologous DNA + LL-37 prime/protein + Montanide boost regimen (group 2) could significantly generate strong immune responses as compared to groups immunized with the DNA constructs (groups 3 & 4). Moreover, immunization of mice with the homologous DNA + LL-37 regimen in low dose of DNA (5 µg) could induce higher immune responses than the homologous naked DNA regimen in high dose of DNA (50 µg) indicating the role of LL-37 as a cell penetrating peptide. Additionally, the heterologous DNA + LL-37 prime/protein + Montanide boost regimen (group 2) induced significantly IFN-gamma secretion from virion-exposed lymphocytes in vitro. CONCLUSION: Generally, the use of LL-37 for DNA delivery, Montanide 720 as an adjuvant, and heterologous DNA prime/protein boost strategy could significantly increase IgG2a, IFN-gamma, and Granzyme B, and maintain cytokine secretion after exposure to virions. Indeed, the heterologous DNA + LL-37 prime/protein + Montanide boost regimen can be considered as a potent strategy for development of therapeutic HIV vaccines.

Tubular-specific expression of HIV protein Vpr leads to severe tubulointerstitial damage accompanied by progressive fibrosis and cystic development.

Chronic kidney disease (CKD) is a common cause of morbidity in human immunodeficiency virus (HIV)-positive individuals. HIV infection leads to a wide spectrum of kidney cell damage, including tubular epithelial cell (TEC) injury. Among the HIV-1 proteins, the pathologic effects of viral protein R (Vpr) are well established and include DNA damage response, cell cycle arrest, and cell death. Several in vitro studies have unraveled the molecular pathways driving the cytopathic effects of Vpr in tubular epithelial cells. However, the in vivo effects of Vpr on tubular injury and CKD pathogenesis have not been thoroughly investigated. Here, we use a novel inducible tubular epithelial cell-specific Vpr transgenic mouse model to show that Vpr expression leads to progressive tubulointerstitial damage, interstitial inflammation and fibrosis, and tubular cyst development. Importantly, Vpr-expressing tubular epithelial cells displayed significant hypertrophy, aberrant cell division, and atrophy; all reminiscent of tubular injuries observed in human HIV-associated nephropathy (HIVAN). Single-cell RNA sequencing analysis revealed the Vpr-mediated transcriptomic responses in specific tubular subsets and highlighted the potential multifaceted role of p53 in the regulation of cell metabolism, proliferation, and death pathways in Vpr-expressing tubular epithelial cells. Thus, our study demonstrates that HIV Vpr expression in tubular cells is sufficient to induce HIVAN-like tubulointerstitial damage and fibrosis, independent of glomerulosclerosis and proteinuria. Additionally, as this new mouse model develops progressive CKD with diffuse fibrosis and kidney failure, it can serve as a useful tool to examine the mechanisms of kidney disease progression and fibrosis in vivo.

Host KIR/HLA-C Genotypes Determine HIV-Mediated Changes of the NK Cell Repertoire and Are Associated With Vpu Sequence Variations Impacting Downmodulation of HLA-C.

NK cells play a pivotal role in viral immunity, utilizing a large array of activating and inhibitory receptors to identify and eliminate virus-infected cells. Killer-cell immunoglobulin-like receptors (KIRs) represent a highly polymorphic receptor family, regulating NK cell activity and determining the ability to recognize target cells. Human leukocyte antigen (HLA) class I molecules serve as the primary ligand for KIRs. Herein, HLA-C stands out as being the dominant ligand for the majority of KIRs. Accumulating evidence indicated that interactions between HLA-C and its inhibitory KIR2DL receptors (KIR2DL1/L2/L3) can drive HIV-1-mediated immune evasion and thus may contribute to the intrinsic control of HIV-1 infection. Of particular interest in this context is the recent observation that HIV-1 is able to adapt to host HLA-C genotypes through Vpu-mediated downmodulation of HLA-C. However, our understanding of the complex interplay between KIR/HLA immunogenetics, NK cell-mediated immune pressure and HIV-1 immune escape is still limited. Therefore, we investigated the impact of specific KIR/HLA-C combinations on the NK cell receptor repertoire and HIV-1 Vpu protein sequence variations of 122 viremic, untreated HIV-1+ individuals. Compared to 60 HIV-1- controls, HIV-1 infection was associated with significant changes within the NK cell receptor repertoire, including reduced percentages of NK cells expressing NKG2A, CD8, and KIR2DS4. In contrast, the NKG2C+ and KIR3DL2+ NK cell sub-populations from HIV-1+ individuals was enlarged compared to HIV-1- controls. Stratification along KIR/HLA-C genotypes revealed a genotype-dependent expansion of KIR2DL1+ NK cells that was ultimately associated with increased binding affinities between KIR2DL1 and HLA-C allotypes. Lastly, our data hinted to a preferential selection of Vpu sequence variants that were associated with HLA-C downmodulation in individuals with high KIR2DL/HLA-C binding affinities. Altogether, our study provides evidence that HIV-1-associated changes in the KIR repertoire of NK cells are to some extent predetermined by host KIR2DL/HLA-C genotypes. Furthermore, analysis of Vpu sequence polymorphisms indicates that differential KIR2DL/HLA-C binding affinities may serve as an additional mechanism how host genetics impact immune evasion by HIV-1.

Dual Role of HIV-1 Envelope Signal Peptide in Immune Evasion.

HIV-1 Env signal peptide (SP) is an important contributor to Env functions. Env is generated from Vpu/Env encoded bicistronic mRNA such that the 5' end of Env-N-terminus, that encodes for Env-SP overlaps with 3' end of Vpu. Env SP displays high sequence diversity, which translates into high variability in Vpu sequence. This study aimed to understand the effect of sequence polymorphism in the Vpu-Env overlapping region (VEOR) on the functions of two vital viral proteins: Vpu and Env. We used infectious molecular clone pNL4.3-CMU06 and swapped its SP (or VEOR) with that from other HIV-1 isolates. Swapping VEOR did not affect virus production in the absence of tetherin however, presence of tetherin significantly altered the release of virus progeny. VEOR also altered Vpu's ability to downregulate CD4 and tetherin. We next tested the effect of these swaps on Env functions. Analyzing the binding of monoclonal antibodies to membrane embedded Env revealed changes in the antigenic landscape of swapped Envs. These swaps affected the oligosaccharide composition of Env-N-glycans as shown by changes in DC-SIGN-mediated virus transmission. Our study suggests that genetic diversity in VEOR plays an important role in the differential pathogenesis and also assist in immune evasion by altering Env epitope exposure.

Novel Compound Inhibitors of HIV-1NL4-3 Vpu.

HIV-1 Vpu targets the host cell proteins CD4 and BST-2/Tetherin for degradation, ultimately resulting in enhanced virus spread and host immune evasion. The discovery and characterization of small molecules that antagonize Vpu would further elucidate the contribution of Vpu to pathogenesis and lay the foundation for the study of a new class of novel HIV-1 therapeutics. To identify novel compounds that block Vpu activity, we have developed a cell-based 'gain of function' assay that produces a positive signal in response to Vpu inhibition. To develop this assay, we took advantage of the viral glycoprotein, GaLV Env. In the presence of Vpu, GaLV Env is not incorporated into viral particles, resulting in non-infectious virions. Vpu inhibition restores infectious particle production. Using this assay, a high throughput screen of >650,000 compounds was performed to identify inhibitors that block the biological activity of Vpu. From this screen, we identified several positive hits but focused on two compounds from one structural family, SRI-41897 and SRI-42371. We developed independent counter-screens for off target interactions of the compounds and found no off target interactions. Additionally, these compounds block Vpu-mediated modulation of CD4, BST-2/Tetherin and antibody dependent cell-mediated toxicity (ADCC). Unfortunately, both SRI-41897 and SRI-42371 were shown to be specific to the N-terminal region of NL4-3 Vpu and did not function against other, more clinically relevant, strains of Vpu; however, this assay may be slightly modified to include more significant Vpu strains in the future.

Combined noncanonical NF-κB agonism and targeted BET bromodomain inhibition reverse HIV latency ex vivo.

Latency reversal strategies for HIV cure using inhibitor of apoptosis protein (IAP) antagonists (IAPi) induce unprecedented levels of latent reservoir expression without immunotoxicity during suppressive antiretroviral therapy (ART). However, full targeting of the reservoir may require combinatorial approaches. A Jurkat latency model screen for IAPi combination partners demonstrated synergistic latency reversal with bromodomain (BD) and extraterminal domain protein inhibitors (BETi). Mechanistic investigations using CRISPR-CAS9 and single-cell RNA-Seq informed comprehensive ex vivo evaluations of IAPi plus pan-BET, bD-selective BET, or selective BET isoform targeting in CD4+ T cells from ART-suppressed donors. IAPi+BETi treatment resulted in striking induction of cell-associated HIV gag RNA, but lesser induction of fully elongated and tat-rev RNA compared with T cell activation-positive controls. IAPi+BETi resulted in HIV protein induction in bulk cultures of CD4+ T cells using an ultrasensitive p24 assay, but did not result in enhanced viral outgrowth frequency using a standard quantitative viral outgrowth assay. This study defines HIV transcriptional elongation and splicing as important barriers to latent HIV protein expression following latency reversal, delineates the roles of BET proteins and their BDs in HIV latency, and provides a rationale for exploration of IAPi+BETi in animal models of HIV latency.

Detecting Selection in the HIV-1 Genome during Sexual Transmission Events.

Little is known about whether and how variation in the HIV-1 genome affects its transmissibility. Assessing which genomic features of HIV-1 are under positive or negative selection during transmission is challenging, because very few virus particles are typically transmitted, and random genetic drift can dilute genetic signals in the recipient virus population. We analyzed 30 transmitter-recipient pairs from the Zurich Primary HIV Infection Study and the Swiss HIV Cohort Study using near full-length HIV-1 genomes. We developed a new statistical test to detect selection during transmission, called Selection Test in Transmission (SeTesT), based on comparing the transmitter and recipient virus population and accounting for the transmission bottleneck. We performed extensive simulations and found that sensitivity of detecting selection during transmission is limited by the strong population bottleneck of few transmitted virions. When pooling individual test results across patients, we found two candidate HIV-1 genomic features for affecting transmission, namely amino acid positions 3 and 18 of Vpu, which were significant before but not after correction for multiple testing. In summary, SeTesT provides a general framework for detecting selection based on genomic sequencing data of transmitted viruses. Our study shows that a higher number of transmitter-recipient pairs is required to improve sensitivity of detecting selection.

Extending the Coding Potential of Viral Genomes with Overlapping Antisense ORFs: A Case for the De Novo Creation of the Gene Encoding the Antisense Protein ASP of HIV-1.

Gene overprinting occurs when point mutations within a genomic region with an existing coding sequence create a new one in another reading frame. This process is quite frequent in viral genomes either to maximize the amount of information that they encode or in response to strong selective pressure. The most frequent scenario involves two different reading frames in the same DNA strand (sense overlap). Much less frequent are cases of overlapping genes that are encoded on opposite DNA strands (antisense overlap). One such example is the antisense ORF, asp in the minus strand of the HIV-1 genome overlapping the env gene. The asp gene is highly conserved in pandemic HIV-1 strains of group M, and it is absent in non-pandemic HIV-1 groups, HIV-2, and lentiviruses infecting non-human primates, suggesting that the ~190-amino acid protein that is expressed from this gene (ASP) may play a role in virus spread. While the function of ASP in the virus life cycle remains to be elucidated, mounting evidence from several research groups indicates that ASP is expressed in vivo. There are two alternative hypotheses that could be envisioned to explain the origin of the asp ORF. On one hand, asp may have originally been present in the ancestor of contemporary lentiviruses, and subsequently lost in all descendants except for most HIV-1 strains of group M due to selective advantage. Alternatively, the asp ORF may have originated very recently with the emergence of group M HIV-1 strains from SIVcpz. Here, we used a combination of computational and statistical approaches to study the genomic region of env in primate lentiviruses to shed light on the origin, structure, and sequence evolution of the asp ORF. The results emerging from our studies support the hypothesis of a recent de novo addition of the antisense ORF to the HIV-1 genome through a process that entailed progressive removal of existing internal stop codons from SIV strains to HIV-1 strains of group M, and fine tuning of the codon sequence in env that reduced the chances of new stop codons occurring in asp. Altogether, the study supports the notion that the HIV-1 asp gene encodes an accessory protein, providing a selective advantage to the virus.

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.

HIV-Proteins-Associated CNS Neurotoxicity, Their Mediators, and Alternative Treatments.

Human immunodeficiency virus (HIV)-infected people's livelihoods are gradually being prolonged with the use of combined antiretroviral therapy (ART). Conversely, despite viral suppression by ART, the symptoms of HIV-associated neurocognitive disorder (HAND) endure. HAND persists because ART cannot really permanently confiscate the virus from the body. HAND encompasses a variety of conditions based on clinical presentation and severity level, comprising asymptomatic neurocognitive impairment, moderate neurocognitive disorder, and HIV-associated dementia. During the early stages of HIV infection, inflammation compromises the blood-brain barrier, allowing toxic virus, infected monocytes, macrophages, T-lymphocytes, and cellular products from the bloodstream to enter the brain and eventually the entire central nervous system. Since there are no resident T-lymphocytes in the brain, the virus will live for decades in macrophages and astrocytes, establishing a reservoir of infection. The HIV proteins then inflame neurons both directly and indirectly. The purpose of this review is to provide a synopsis of the effects of these proteins on the central nervous system and conceptualize avenues to be considered in mitigating HAND. We used bioinformatics repositories extensively to simulate the transcription factors that bind to the promoter of the HIV-1 protein and possibly could be used as a target to circumvent HIV-associated neurocognitive disorders. In the same vein, a protein-protein interaction complex was also deduced from a Search Tool for the Retrieval of Interacting Genes. In conclusion, this provides an alternative strategy that could be used to avert HAND.

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.

Unravelling the Immunomodulatory Effects of Viral Ion Channels, towards the Treatment of Disease.

The current COVID-19 pandemic has highlighted the need for the research community to develop a better understanding of viruses, in particular their modes of infection and replicative lifecycles, to aid in the development of novel vaccines and much needed anti-viral therapeutics. Several viruses express proteins capable of forming pores in host cellular membranes, termed "Viroporins". They are a family of small hydrophobic proteins, with at least one amphipathic domain, which characteristically form oligomeric structures with central hydrophilic domains. Consequently, they can facilitate the transport of ions through the hydrophilic core. Viroporins localise to host membranes such as the endoplasmic reticulum and regulate ion homeostasis creating a favourable environment for viral infection. Viroporins also contribute to viral immune evasion via several mechanisms. Given that viroporins are often essential for virion assembly and egress, and as their structural features tend to be evolutionarily conserved, they are attractive targets for anti-viral therapeutics. This review discusses the current knowledge of several viroporins, namely Influenza A virus (IAV) M2, Human Immunodeficiency Virus (HIV)-1 Viral protein U (Vpu), Hepatitis C Virus (HCV) p7, Human Papillomavirus (HPV)-16 E5, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Open Reading Frame (ORF)3a and Polyomavirus agnoprotein. We highlight the intricate but broad immunomodulatory effects of these viroporins and discuss the current antiviral therapies that target them; continually highlighting the need for future investigations to focus on novel therapeutics in the treatment of existing and future emergent viruses.

Retroviral Antisense Transcripts and Genes: 33 Years after First Predicted, a Silent Retroviral Revolution?

Paradigm shifts throughout the history of microbiology have typically been ignored, or met with skepticism and resistance, by the scientific community. This has been especially true in the field of virology, where the discovery of a "contagium vivum fluidum", or infectious fluid remaining after excluding bacteria by filtration, was initially ignored because it did not coincide with the established view of microorganisms. Subsequent studies on such infectious agents, eventually termed "viruses", were met with skepticism. However, after an abundance of proof accumulated, viruses were eventually acknowledged as defined microbiological entities. Next, the proposed role of viruses in oncogenesis in animals was disputed, as was the unique mechanism of genome replication by reverse transcription of RNA by the retroviruses. This same pattern of skepticism holds true for the prediction of the existence of retroviral "antisense" transcripts and genes. From the time of their discovery, it was thought that retroviruses encoded proteins on only one strand of proviral DNA. However, in 1988, it was predicted that human immunodeficiency virus type 1 (HIV-1), and other retroviruses, express an antisense protein encoded on the DNA strand opposite that encoding the known viral proteins. Confirmation came quickly with the characterization of the antisense protein, HBZ, of the human T-cell leukemia virus type 1 (HTLV-1), and the finding that both the protein and its antisense mRNA transcript play key roles in viral replication and pathogenesis. However, acceptance of the existence, and potential importance, of a corresponding antisense transcript and protein (ASP) in HIV-1 infection and pathogenesis has lagged, despite gradually accumulating theoretical and experimental evidence. The most striking theoretical evidence is the finding that asp is highly conserved in group M viruses and correlates exclusively with subtypes, or clades, responsible for the AIDS pandemic. This review outlines the history of the major shifts in thought pertaining to the nature and characteristics of viruses, and in particular retroviruses, and details the development of the hypothesis that retroviral antisense transcripts and genes exist. We conclude that there is a need to accelerate studies on ASP, and its transcript(s), with the view that both may be important, and overlooked, targets in anti-HIV therapeutic and vaccine strategies.