Low back pain and osteoarthritis pain: a perspective of estrogen.

Low back pain (LBP) is the world's leading cause of disability and is increasing in prevalence more rapidly than any other pain condition. Intervertebral disc (IVD) degeneration and facet joint osteoarthritis (FJOA) are two common causes of LBP, and both occur more frequently in elderly women than in other populations. Moreover, osteoarthritis (OA) and OA pain, regardless of the joint, are experienced by up to twice as many women as men, and this difference is amplified during menopause. Changes in estrogen may be an important contributor to these pain states. Receptors for estrogen have been found within IVD tissue and nearby joints, highlighting the potential roles of estrogen within and surrounding the IVDs and joints. In addition, estrogen supplementation has been shown to be effective at ameliorating IVD degeneration and OA progression, indicating its potential use as a therapeutic agent for people with LBP and OA pain. This review comprehensively examines the relationship between estrogen and these pain conditions by summarizing recent preclinical and clinical findings. The potential molecular mechanisms by which estrogen may relieve LBP associated with IVD degeneration and FJOA and OA pain are discussed.

Defective Interfering Particles with Broad-Acting Antiviral Activity for Dengue, Zika, Yellow Fever, Respiratory Syncytial and SARS-CoV-2 Virus Infection.

More than 100 arboviruses, almost all of which have an RNA genome, cause disease in humans. RNA viruses are causing unprecedented health system challenges worldwide, many with little or no specific therapies or vaccines available. Certain species of mosquito can carry dengue virus (DENV), Zika virus (ZIKV) and yellow fever virus (YFV), where co-infection of these viruses has occurred. Here, we found that purified synthetic defective interfering particles (DIPs) derived from DENV type 2 (DENV-2) strongly suppressed replication of the aforementioned viruses, respiratory syncytial virus (RSV) and also the novel emerging virus SARS-CoV-2 in human cells. DENV DIPs produced in bioreactors, purified by column chromatography, and concentrated are virus-like particles that are about half the diameter of a typical DENV particle, but with similar ratios of the viral structural proteins envelope and capsid. Overall, DIP-treated cells inhibited DENV, ZIKV, YFV, RSV, and SARS-CoV-2 by at least 98% by mechanisms which included interferon (IFN)-dependent cellular antiviral responses. IMPORTANCE DIPs are spontaneously derived virus mutants with deletions in genes that block viral replication. DIPs play important roles in modulation of viral disease, innate immune responses, virus persistence and virus evolution. Here, we investigated the antiviral activity of highly purified synthetic DIPs derived from DENV, which were produced in bioreactors. DENV DIPs purified by column chromatography strongly inhibited five different RNA viruses, including DENV, ZIKV, YFV, RSV, and SARS-CoV-2 in human cells. DENV DIPs inhibited virus replication via delivery of a small, noninfectious viral RNA that activated cellular innate immunity, resulting in robust type 1 interferon responses. The work here presents a pathway for DIP production which is adaptable to Good Manufacturing Practice, so that their preclinical testing should be suitable for evaluation in subjects.

Dengue virus-free defective interfering particles have potent and broad anti-dengue virus activity.

Dengue virus (DENV) is spread from human to human through the bite of the female Aedes aegypti mosquito and leads to about 100 million clinical infections yearly. Treatment options and vaccine availability for DENV are limited. Defective interfering particles (DIPs) are considered a promising antiviral approach but infectious virus contamination has limited their development. Here, a DENV-derived DIP production cell line was developed that continuously produced DENV-free DIPs. The DIPs contained and could deliver to cells a DENV serotype 2 subgenomic defective-interfering RNA, which was originally discovered in DENV infected patients. The DIPs released into cell culture supernatant were purified and could potently inhibit replication of all DENV serotypes in cells. Antiviral therapeutics are limited for many viral infection. The DIP system described could be re-purposed to make antiviral DIPs for many other RNA viruses such as SARS-CoV-2, yellow fever, West Nile and Zika viruses.

The unique features of SARS-CoV-2 transmission: Comparison with SARS-CoV, MERS-CoV and 2009 H1N1 pandemic influenza virus.

From 2002 to 2019, three deadly human coronaviruses (hCoVs), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle Eastern respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged to produce outbreaks of SARS, MERS and coronavirus disease 2019 (Covid-19), respectively. All three hCoVs are members of the Betacoronavirus genus in the subfamily Orthocoronavirinae and share many similarities in virology and epidemiology. However, the pattern and scale of Covid-19 global spread is similar to 2009 pandemic H1N1 influenza (H1N1pdm09), rather than SARS or MERS. Covid-19 exhibits high viral shedding in the upper respiratory tract at an early stage of infection, and has a high proportion of transmission competent individuals that are pre-symptomatic, asymptomatic and mildly symptomatic, characteristics seen in H1N1pdm09 but not in SARS or MERS. These two traits of Covid-19 and H1N1pdm09 result in reduced efficiency in identification of transmission sources by symptomatic screening and play important roles in their ability to spread unchecked to cause pandemics. To overcome these attributes of Covid-19 in community transmission, identifying the transmission source by testing for virus shedding and interrupting chains of transmission by social distancing and public masking are required.

Assessing the Binding of Venoms from Aquatic Elapids to the Nicotinic Acetylcholine Receptor Orthosteric Site of Different Prey Models.

The evolution of an aquatic lifestyle from land dwelling venomous elapids is a radical ecological modification, bringing about many evolutionary changes from morphology to diet. Diet is an important ecological facet which can play a key role in regulating functional traits such as venom composition and prey-specific targeting of venom. In addition to predating upon novel prey (e.g., fish, fish eggs and invertebrates), the venoms of aquatic elapids also face the challenge of increased prey-escape potential in the aquatic environment. Thus, despite the independent radiation into an aquatic niche on four separate occasions, the venoms of aquatic elapids are evolving under convergent selection pressures. Utilising a biolayer interferometry binding assay, this study set out to elucidate whether crude venoms from representative aquatic elapids were target-specific to the orthosteric site of postsynaptic nicotinic acetylcholine receptor mimotopes of fish compared to other terrestrial prey types. Representatives of the four aquatic lineages were: aquatic coral snakes representative was Micrurus surinamensis;, sea kraits representative was Laticauda colubrina; sea snakes representatives were two Aipysurus spp. and eight Hydrophis spp; and water cobras representative was Naja annulata. No prey-specific differences in crude venom binding were observed from any species tested, except for Aipysurus laevis, which showed slight evidence of prey-potency differences. For Hydrophis caerulescens, H. peronii, H. schistosus and M. surinamensis, there was a lack of binding to the orthosteric site of any target lineage. Subsequent testing on the in vitro chick-biventer cervicis muscle preparation suggested that, while the venoms of these species bound postsynaptically, they bound to allosteric sites rather than orthosteric. Allosteric binding is potentially a weaker but faster-acting form of neurotoxicity and we hypothesise that the switch to allosteric binding is likely due to selection pressures related to prey-escape potential. This research has potentially opened up the possibility of a new functional class of toxins which have never been assessed previously while shedding light on the selection pressures shaping venom evolution.

Evolutionary Interpretations of Nicotinic Acetylcholine Receptor Targeting Venom Effects by a Clade of Asian Viperidae Snakes.

Ecological variability among closely related species provides an opportunity for evolutionary comparative studies. Therefore, to investigate the origin and evolution of neurotoxicity in Asian viperid snakes, we tested the venoms of Azemiops feae, Calloselasma rhodostoma, Deinagkistrodon acutus, Tropidolaeums subannulatus, and T. wagleri for their relative specificity and potency upon the amphibian, lizard, bird, rodent, and human α-1 (neuromuscular) nicotinic acetylcholine receptors. We utilised a biolayer interferometry assay to test the binding affinity of these pit viper venoms to orthosteric mimotopes of nicotinic acetylcholine receptors binding region from a diversity of potential prey types. The Tropidolaemus venoms were much more potent than the other species tested, which is consistent with the greater prey escape potential in arboreal niches. Intriguingly, the venom of C. rhodostoma showed neurotoxic binding to the α-1 mimotopes, a feature not known previously for this species. The lack of prior knowledge of neurotoxicity in this species is consistent with our results due to the bias in rodent studies and human bite reports, whilst this venom had a greater binding affinity toward amphibian and diapsid α-1 targets. The other large terrestrial species, D. acutus, did not display any meaningful levels of neurotoxicity. These results demonstrate that whilst small peptide neurotoxins are a basal trait of these snakes, it has been independently amplified on two separate occasions, once in Azemiops and again in Tropidolaemus, and with Calloselasma representing a third possible amplification of this trait. These results also point to broader sources of novel neuroactive peptides with the potential for use as lead compounds in drug design and discovery.

Tat-Based Therapies as an Adjuvant for an HIV-1 Functional Cure.

The human immunodeficiency virus type 1 (HIV) establishes a chronic infection that can be well controlled, but not cured, by combined antiretroviral therapy (cART). Interventions have been explored to accomplish a functional cure, meaning that a patient remains infected but HIV is undetectable in the blood, with the aim of allowing patients to live without cART. Tat, the viral transactivator of transcription protein, plays a critical role in controlling HIV transcription, latency, and viral rebound following the interruption of cART treatment. Therefore, a logical approach for controlling HIV would be to block Tat. Tackling Tat with inhibitors has been a difficult task, but some recent discoveries hold promise. Two anti-HIV proteins, Nullbasic (a mutant of Tat) and HT1 (a fusion of HEXIM1 and Tat functional domains) inhibit viral transcription by interfering with the interaction of Tat and cellular factors. Two small molecules, didehydro-cortistatin A (dCA) and triptolide, inhibit Tat by different mechanisms: dCA through direct binding and triptolide through enhanced proteasomal degradation. Finally, two Tat-based vaccines under development elicit Tat-neutralizing antibodies. These vaccines have increased the levels of CD4+ cells and reduced viral loads in HIV-infected people, suggesting that the new vaccines are therapeutic. This review summarizes recent developments of anti-Tat agents and how they could contribute to a functional cure for HIV.

An Appetite for Destruction: Detecting Prey-Selective Binding of α-Neurotoxins in the Venom of Afro-Asian Elapids.

Prey-selective venoms and toxins have been documented across only a few species of snakes. The lack of research in this area has been due to the absence of suitably flexible testing platforms. In order to test more species for prey specificity of their venom, we used an innovative taxonomically flexible, high-throughput biolayer interferometry approach to ascertain the relative binding of 29 α-neurotoxic venoms from African and Asian elapid representatives (26 Naja spp., Aspidelaps scutatus, Elapsoidea boulengeri, and four locales of Ophiophagus hannah) to the alpha-1 nicotinic acetylcholine receptor orthosteric (active) site for amphibian, lizard, snake, bird, and rodent targets. Our results detected prey-selective, intraspecific, and geographical differences of α-neurotoxic binding. The results also suggest that crude venom that shows prey selectivity is likely driven by the proportions of prey-specific α-neurotoxins with differential selectivity within the crude venom. Our results also suggest that since the α-neurotoxic prey targeting does not always account for the full dietary breadth of a species, other toxin classes with a different pathophysiological function likely play an equally important role in prey immobilisation of the crude venom depending on the prey type envenomated. The use of this innovative and taxonomically flexible diverse assay in functional venom testing can be key in attempting to understanding the evolution and ecology of α-neurotoxic snake venoms, as well as opening up biochemical and pharmacological avenues to explore other venom effects.

Assessing the binding of venoms from aquatic elapids to the nicotinic acetylcholine receptor orthosteric site of different prey models

The evolution of an aquatic lifestyle from land dwelling venomous elapids is a radical ecological modification, bringing about many evolutionary changes from morphology to diet. Diet is an important ecological facet which can play a key role in regulating functional traits such as venom composition and prey-specific targeting of venom. In addition to predating upon novel prey (e.g., fish, fish eggs and invertebrates), the venoms of aquatic elapids also face the challenge of increased prey-escape potential in the aquatic environment. Thus, despite the independent radiation into an aquatic niche on four separate occasions, the venoms of aquatic elapids are evolving under convergent selection pressures. Utilising a biolayer interferometry binding assay, this study set out to elucidate whether crude venoms from representative aquatic elapids were target-specific to the orthosteric site of postsynaptic nicotinic acetylcholine receptor mimotopes of fish compared to other terrestrial prey types. Representatives of the four aquatic lineages were: aquatic coral snakes representative was Micrurus surinamensis;, sea kraits representative was Laticauda colubrina; sea snakes representatives were two Aipysurus spp. and eight Hydrophis spp; and water cobras representative was Naja annulata. No prey-specific differences in crude venom binding were observed from any species tested, except for Aipysurus laevis, which showed slight evidence of prey-potency differences. For Hydrophis caerulescens, H. peronii, H. schistosus and M. surinamensis, there was a lack of binding to the orthosteric site of any target lineage. Subsequent testing on the in vitro chick-biventer cervicis muscle preparation suggested that, while the venoms of these species bound postsynaptically, they bound to allosteric sites rather than orthosteric. Allosteric binding is potentially a weaker but faster-acting form of neurotoxicity and we hypothesise that the switch to allosteric binding is likely due to selection pressures related to prey-escape potential. This research has potentially opened up the possibility of a new functional class of toxins which have never been assessed previously while shedding light on the selection pressures shaping venom evolution.

A Taxon-Specific and High-Throughput Method for Measuring Ligand Binding to Nicotinic Acetylcholine Receptors.

The binding of compounds to nicotinic acetylcholine receptors is of great interest in biomedical research. However, progress in this area is hampered by the lack of a high-throughput, cost-effective, and taxonomically flexible platform. Current methods are low-throughput, consume large quantities of sample, or are taxonomically limited in which targets can be tested. We describe a novel assay which utilizes a label-free bio-layer interferometry technology, in combination with adapted mimotope peptides, in order to measure ligand binding to the orthosteric site of nicotinic acetylcholine receptor alpha-subunits of diverse organisms. We validated the method by testing the evolutionary patterns of a generalist feeding species (Acanthophis antarcticus), a fish specialist species (Aipysurus laevis), and a snake specialist species (Ophiophagus hannah) for comparative binding to the orthosteric site of fish, amphibian, lizard, snake, bird, marsupial, and rodent alpha-1 nicotinic acetylcholine receptors. Binding patterns corresponded with diet, with the Acanthophis antarcticus not showing bias towards any particular lineage, while Aipysurus laevis showed selectivity for fish, and Ophiophagus hannah a selectivity for snake. To validate the biodiscovery potential of this method, we screened Acanthophis antarcticus and Tropidolaemus wagleri venom for binding to human alpha-1, alpha-2, alpha-3, alpha-4, alpha-5, alpha-6, alpha-7, alpha-9, and alpha-10. While A. antarcticus was broadly potent, T. wagleri showed very strong but selective binding, specifically to the alpha-1 target which would be evolutionarily selected for, as well as the alpha-5 target which is of major interest for drug design and development. Thus, we have shown that our novel method is broadly applicable for studies including evolutionary patterns of venom diversification, predicting potential neurotoxic effects in human envenomed patients, and searches for novel ligands of interest for laboratory tools and in drug design and development.

Strong In Vivo Inhibition of HIV-1 Replication by Nullbasic, a Tat Mutant.

Nullbasic is a mutant form of the HIV-1 transcriptional activator protein (Tat) that strongly inhibits HIV-1 transcription and replication in lymphocytes in vitro To investigate Nullbasic inhibition in vivo, we employed an NSG mouse model where animals were engrafted with primary human CD4+ cells expressing a Nullbasic-ZsGreen1 (NB-ZSG) fusion protein or ZSG. NB-ZSG and ZSG were delivered by using a retroviral vector where CD4+ cells were transduced either prior to (preinfection) or following (postinfection) HIV-1 infection. The transduced cells were analyzed in vitro up to 10 days postinfection (dpi) and in vivo up to 39 dpi. Compared to ZSG, NB-ZSG strongly inhibited HIV-1 replication both in vitro and in vivo using preinfection treatment. In vitro, HIV-1 mRNA levels in cells were reduced by up to 60-fold. In vivo, HIV-1 RNA was undetectable in plasma samples during the course of the experiment, and HIV-1 mRNA levels in resident CD4+ cells in organ tissue were reduced up to 2,800-fold. Postinfection treatment of HIV-1-infected cells with NB-ZSG attenuated HIV-1 infection for up to 14 days. In vitro, a 25-fold reduction of viral mRNA in cells was observed but diminished to a <2-fold reduction by 10 dpi. In vivo, HIV-1 RNA was undetectable in plasma of NB-ZSG mice at 14 dpi but afterwards was not significantly different between NB-ZSG mice and control mice. However, we observed higher levels of CD4+ cells in NB-ZSG mice than in control mice, suggesting that NB-ZSG imparted a survival advantage to HIV-1-infected animals.IMPORTANCE HIV-1 infection is effectively controlled by antiviral therapy that inhibits virus replication and reduces viral loads below detectable levels in patients. However, therapy interruption leads to viral rebound due to latently infected cells, which serve as a source of continued viral infection. Interest in strategies leading to a functional cure for HIV-1 infection by long-term or permanent viral suppression is growing. Here, we show that a mutant form of the HIV-1 Tat protein, referred to as Nullbasic, inhibits HIV-1 transcription in infected CD4+ cells in vivo Analysis shows that stable expression of Nullbasic in CD4+ cells could lead to durable anti-HIV-1 activity. Nullbasic, as a gene therapy candidate, could be a part of a functional-cure strategy to suppress HIV-1 transcription and replication.

Oxazole-Benzenesulfonamide Derivatives Inhibit HIV-1 Reverse Transcriptase Interaction with Cellular eEF1A and Reduce Viral Replication.

HIV-1 replication requires direct interaction between HIV-1 reverse transcriptase (RT) and cellular eukaryotic translation elongation factor 1A (eEF1A). Our previous work showed that disrupting this interaction inhibited HIV-1 uncoating, reverse transcription, and replication, indicating its potential as an anti-HIV-1 target. In this study, we developed a sensitive, live-cell split-luciferase complementation assay (NanoBiT) to quantitatively measure inhibition of HIV-1 RT interaction with eEF1A. We used this to screen a small molecule library and discovered small-molecule oxazole-benzenesulfonamides (C7, C8, and C9), which dose dependently and specifically inhibited the HIV-1 RT interaction with eEF1A. These compounds directly bound to HIV-1 RT in a dose-dependent manner, as assessed by a biolayer interferometry (BLI) assay, but did not bind to eEF1A. These oxazole-benzenesulfonamides did not inhibit enzymatic activity of recombinant HIV-1 RT in a homopolymer assay but did inhibit reverse transcription and infection of both wild-type (WT) and nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 in a dose-dependent manner in HEK293T cells. Infection of HeLa cells was significantly inhibited by the oxazole-benzenesulfonamides, and the antiviral activity was most potent against replication stages before 8 h postinfection. In human primary activated CD4+ T cells, C7 inhibited HIV-1 infectivity and replication up to 6 days postinfection. The data suggest a novel mechanism of HIV-1 inhibition and further elucidate how the RT-eEF1A interaction is important for HIV-1 replication. These compounds provide potential to develop a new class of anti-HIV-1 drugs to treat WT and NNRTI-resistant strains in people infected with HIV.IMPORTANCE Antiretroviral drugs protect many HIV-positive people, but their success can be compromised by drug-resistant strains. To combat these strains, the development of new classes of HIV-1 inhibitors is essential and a priority in the field. In this study, we identified small molecules that bind directly to HIV-1 reverse transcriptase (RT) and inhibit its interaction with cellular eEF1A, an interaction which we have previously identified as crucial for HIV-1 replication. These compounds inhibit intracellular HIV-1 reverse transcription and replication of WT HIV-1, as well as HIV-1 mutants that are resistant to current RT inhibitors. A novel mechanism of action involving inhibition of the HIV-1 RT-eEF1A interaction is an important finding and a potential new way to combat drug-resistant HIV-1 strains in infected people.

eEF1A demonstrates paralog specific effects on HIV-1 reverse transcription efficiency.

The eukaryotic translation elongation factor 1A (eEF1A) has two cell-type specific paralogs, eEF1A1 and eEF1A2. Both paralogs undertake a canonical function in delivering aminoacyl-tRNA to the ribosome for translation, but differences in other functions are emerging. eEF1A1 has been reported to be important for the replication of many viruses, but no study has specifically linked the eEF1A2 paralog. We have previously demonstrated that eEF1A1 directly interacts with HIV-1 RT and supports efficient reverse transcription. Here, we showed that RT interacted more strongly with eEF1A1 than with eEF1A2 in immunoprecipitation assay. Biolayer interferometry using eEF1A paralogs showed different association and dissociation rates with RT. Over expressed eEF1A1, but not eEF1A2, was able to restore HIV-1 reverse transcription efficiency in HEK293T cells with endogenous eEF1A knocked-down and HIV-1 reverse transcription efficiency correlated with the level of eEF1A1 mRNA, but not to eEF1A2 mRNA in both HEK293T and primary human skeletal muscle cells.

The eukaryotic translation elongation factor 1A regulation of actin stress fibers is important for infectious RSV production.

Cellular protein eukaryotic translation elongation factor 1A (eEF1A) is an actin binding protein that plays a role in the formation of filamentous actin (F-actin) bundles. F-Actin regulates multiple stages of respiratory syncytial virus (RSV) replication including assembly and budding. Our previous study demonstrated that eEF1A knock-down significantly reduced RSV replication. Here we investigated if the eEF1A function in actin bundle formation was important for RSV replication and release. To investigate this, eEF1A function was impaired in HEp-2 cells by either knock-down of eEF1A with siRNA, or treatment with an eEF1A inhibitor, didemnin B (Did B). Cell staining and confocal microscopy analysis showed that both eEF1A knock-down and treatment with Did B resulted in disruption of cellular stress fiber formation and elevated accumulation of F-actin near the plasma membrane. When treated cells were then infected with RSV, there was also reduced formation of virus-induced cellular filopodia. Did B treatment, similarly to eEF1A knock-down, reduced the release of infectious RSV, but unlike eEF1A knock-down, did not significantly affect RSV genome replication. The lower infectious virus production in Did B treated cells also reduced RSV-induced cell death. In conclusion, the cellular factor eEF1A plays an important role in the regulation of F-actin stress fiber formation required for RSV assembly and release.

RNA glycosidase and other agents target Tat to inhibit HIV-1 transcription.

The HIV-1 tat gene encodes a small 86-104 amino acid protein depending on the HIV-1 strain. Tat is essential for HIV-1 replication through interactions with numerous cellular transcription factors. The interaction between Tat and P-TEFb, which is a cellular protein complex composed of cyclin T1 and CDK9, delivers P-TEFb to the newly transcribed viral mRNAs where phosphorylation of RNA polymerase II by CDK9 leads to highly efficient mRNA transcription. It has long been recognized that Tat is a potential anti-HIV-1 target and possibly a viral Achilles' heel. However, specifically targeting Tat without affecting normal host cell functions has been challenging. Means to inactivate Tat have been reported that includes small compounds, transdominant negative Tat proteins, and by plant-derived antivirals. Investigations of these agents have reported encouraging outcomes that inform and may hopefully affect strategies for a functional HIV-1 cure.

Differential Effects of Strategies to Improve the Transduction Efficiency of Lentiviral Vector that Conveys an Anti-HIV Protein, Nullbasic, in Human T Cells.

Nullbasic is a mutant form of HIV-1 Tat that has strong ability to protect cells from HIV-1 replication by inhibiting three different steps of viral replication: reverse transcription, Rev export of viral mRNA from the nucleus to the cytoplasm and transcription of viral mRNA by RNA polymerase II. We previously showed that Nullbasic inhibits transduction of human cells including T cells by HIV-1-based lentiviral vectors. Here we investigated whether the Nullbasic antagonists huTat2 (a Tat targeting intrabody), HIV-1 Tat or Rev proteins or cellular DDX1 protein could improve transduction by a HIV-1 lentiviral vector conveying Nullbasic-ZsGreen1 to human T cells. We show that overexpression of huTat2, Tat-FLAG and DDX1-HA in virus-like particle (VLP) producer cells significantly improved transduction efficiency of VLPs that convey Nullbasic in Jurkat cells. Specifically, co-expression of Tat-FLAG and DDX1-HA in the VLP producer cell improved transduction efficiency better than if used individually. Transduction efficiencies could be further improved by including a spinoculation step. However, the same optimised protocol and using the same VLPs failed to transduce primary human CD4+ T cells. The results imply that the effects of Nullbasic on VLPs on early HIV-1 replication are robust in human CD4+ T cells. Given this significant block to lentiviral vector transduction by Nullbasic in primary CD4+ T cells, our data indicate that gammaretroviral, but not lentiviral, vectors are suitable for delivering Nullbasic to primary human T cells.

HIV-1 Uncoating and Reverse Transcription Require eEF1A Binding to Surface-Exposed Acidic Residues of the Reverse Transcriptase Thumb Domain.

Once HIV-1 enters a cell, the viral core is uncoated by a poorly understood mechanism and the HIV-1 genomic RNA is reverse transcribed into DNA. Host cell factors are essential for these processes, although very few reverse transcription complex binding host cell factors have been convincingly shown to affect uncoating or reverse transcription. We previously reported that cellular eukaryotic translation elongation factor 1A (eEF1A) interacts tightly and directly with HIV-1 reverse transcriptase (RT) for more efficient reverse transcription. Here we report that the surface-exposed acidic residues in the HIV-1 RT thumb domain alpha-J helix and flanking regions are important for interaction with eEF1A. Mutation of surface-exposed acidic thumb domain residues D250, E297, E298, and E300 to arginine resulted in various levels of impairment of the interaction between RT and eEF1A. This indicates that this negatively charged region in the RT thumb domain is important for interaction with the positively charged eEF1A protein. The impairment of RT and eEF1A interaction by the RT mutations correlated with the efficiency of reverse transcription, uncoating, and infectivity. The best example of this is the strictly conserved E300 residue, where mutation significantly impaired the interaction of RT with eEF1A and virus replication in CD4+ T cells without affecting in vitro RT catalytic activity, RT heterodimerization, or RNase H activity. This study demonstrated that the interaction between surface-exposed acidic residues of the RT thumb domain and eEF1A is important for HIV-1 uncoating, reverse transcription, and replication.IMPORTANCE HIV-1, like all viruses, requires host cell proteins for its replication. Understanding the mechanisms behind virus-host interactions can lay the foundation for future novel therapeutic developments. Our lab has identified eEF1A as a key HIV-1 RT binding host protein that is important for the reverse transcription of HIV-1 genomic RNA into DNA. Here we identify the first surface-exposed RT residues that underpin interactions with eEF1A. Mutation of one strictly conserved RT residue (E300R) delayed reverse transcription and viral core uncoating and strongly inhibited HIV-1 replication in CD4+ T cells. This study advances the structural and mechanistic detail of the key RT-eEF1A interaction in HIV-1 infection and indicates its importance in uncoating for the first time. This provides a further basis for the development of an RT-eEF1A interaction-inhibiting anti-HIV-1 drug and suggests that the surface-exposed acidic patch of the RT thumb domain may be an attractive drug target.

A mutant Tat protein inhibits infection of human cells by strains from diverse HIV-1 subtypes.

BACKGROUND: Nullbasic is a mutant HIV-1 Tat protein that inhibits HIV-1 replication via three independent mechanisms that disrupts 1) reverse transcription of the viral RNA genome into a DNA copy, 2) HIV-1 Rev protein function required for viral mRNA transport from the nucleus to the cytoplasm and 3) HIV-1 mRNA transcription by RNA Polymerase II. The Nullbasic protein is derived from the subtype B strain HIV-1BH10 and has only been tested against other HIV-1 subtype B strains. However, subtype B strains only account for ~10% of HIV-1 infections globally and HIV-1 Tat sequences vary between subtypes especially for subtype C, which is responsible for ~50% HIV-1 infection worldwide. These differences could influence the ability of Tat to interact with RNA and cellular proteins and thus could affect the antiviral activity of Nullbasic. Therefore, Nullbasic was tested against representative HIV-1 strains from subtypes C, D and A/D recombinant to determine if it can inhibit their replication. METHODS: Nullbasic was delivered to human cells using a self-inactivating (SIN) γ-retroviral system. We evaluated Nullbasic-mCherry (NB-mCh) fusion protein activity against the HIV-1 strains in TZM-bl cell lines for inhibition of transactivation and virus replication. We also examined antiviral activity of Nullbasic-ZsGreen1 (NB-ZSG1) fusion protein against the same strains in primary CD4+ T cells. The Nullbasic expression was monitored by western blot and flow cytometry. The effects of Nullbasic on primary CD4+ T cells cytotoxicity, proliferation and apoptosis were also examined. RESULTS: The results show that Nullbasic inhibits Tat-mediated transactivation and virus replication of all the HIV-1 strains tested in TZM-bl cells. Importantly, Nullbasic inhibits replication of the HIV-1 strains in primary CD4+ T cells without affecting cell proliferation, cytotoxicity or level of apoptotic cells. CONCLUSION: A SIN-based γ-retroviral vector used to express Nullbasic fusion proteins improved protein expression particularly in primary CD4+ T cells. Nullbasic has antiviral activity against all strains from the subtypes tested although small differences in viral inhibition were observed. Further improvement of in γ-retroviral vector stable expression of Nullbasic expression may have utility in a future gene therapy approach applicable to genetically diverse HIV-1 strains.

Shutdown of HIV-1 Transcription in T Cells by Nullbasic, a Mutant Tat Protein.

UNLABELLED: Nullbasic is a derivative of the HIV-1 transactivator of transcription (Tat) protein that strongly inhibits HIV-1 replication in lymphocytes. Here we show that lentiviral vectors that constitutively express a Nullbasic-ZsGreen1 (NB-ZSG1) fusion protein by the eEF1α promoter led to robust long-term inhibition of HIV-1 replication in Jurkat cells. Although Jurkat-NB-ZSG1 cells were infected by HIV-1, no virus production could be detected and addition of phorbol ester 12-myristate 13-acetate (PMA) and JQ1 had no effect, while suberanilohydroxamic acid (SAHA) modestly stimulated virus production but at levels 300-fold lower than those seen in HIV-1-infected Jurkat-ZSG1 cells. Virus replication was not recovered by coculture of HIV-1-infected Jurkat-NB-ZSG1 cells with uninfected Jurkat cells. Latently infected Jurkat latent 6.3 and ACH2 cells treated with latency-reversing agents produced measurable viral capsid (CA), but little or none was made when they expressed NB-ZSG1. When Jurkat cells chronically infected with HIV-1 were transduced with lentiviral virus-like particles conveying NB-ZSG1, a >3-log reduction in CA production was observed. Addition of PMA increased virus CA production but at levels 500-fold lower than those seen in nontransduced Jurkat cells. Transcriptome sequencing analysis confirmed that HIV-1 mRNA was strongly inhibited by NB-ZSG1 but indicated that full-length viral mRNA was made. Analysis of HIV-1-infected Jurkat cells expressing NB-ZSG1 by chromatin immunoprecipitation assays indicated that recruitment of RNA polymerase II (RNAPII) and histone 3 lysine 9 acetylation were inhibited. The reduction of HIV-1 promoter-associated RNAPII and epigenetic changes in viral nucleosomes indicate that Nullbasic can inhibit HIV-1 replication by enforcing viral silencing in cells. IMPORTANCE: HIV-1 infection is effectively controlled by antiviral therapy that inhibits virus replication and reduces measurable viral loads in patients below detectable levels. However, therapy interruption leads to viral rebound due to latently infected cells that serve as a source of continued viral infection. Interest in strategies leading to a functional cure of HIV infection by permanent viral suppression, which may be achievable, is growing. Here we show that a mutant form of the HIV-1 Tat protein, referred to as Nullbasic, can inhibit HIV-1 transcription in infected Jurkat T cell to undetectable levels. Analysis shows that Nullbasic alters the epigenetic state of the HIV-1 long terminal repeat promoter, inhibiting its association with RNA polymerase II. This study indicates that key cellular proteins and pathways targeted here can silence HIV-1 transcription. Further elucidation could lead to functional-cure strategies by suppression of HIV transcription, which may be achievable by a pharmacological method.