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We evaluated the in vivo efficacy of structurally flexible, cationic PAMAM dendrimers as a small interfering RNA (siRNA) delivery system in a Rag2‐/‐γc‐/‐ (RAG-hu) humanized mouse model for HIV-1 infection. HIV-infected humanized Rag2‐/‐γc‐/‐ mice (RAG-hu) were injected intravenously (i.v.) with dendrimer-siRNA nanoparticles consisting of a cocktail of dicer substrate siRNAs (dsiRNAs) targeting both viral and cellular transcripts. We report in this study that the dendrimer-dsiRNA treatment suppressed HIV-1 infection by several orders of magnitude and protected against viral induced CD4+ T-cell depletion. We also demonstrated that follow-up injections of the dendrimer-cocktailed dsiRNAs following viral rebound resulted in complete inhibition of HIV-1 titers. Biodistribution studies demonstrate that the dendrimer-dsiRNAs preferentially accumulate in peripheral blood mononuclear cells (PBMCs) and liver and do not exhibit any discernable toxicity. These data demonstrate for the first time efficacious combinatorial delivery of anti-host and -viral siRNAs for HIV-1 treatment in vivo. The dendrimer delivery approach therefore represents a promising method for systemic delivery of combinations of siRNAs for treatment of HIV-1 infection.

Lentiviral vectors are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the lentiviral vector components are cytotoxic, transient plasmid transfection has been used to produce the large batches needed for clinical trials. However, this method is costly, poorly reproducible and hard to scale up. Here we describe a general method for construction of stable packaging cell lines that continuously produce lentiviral vectors. This uses Cre recombinase-mediated cassette exchange to insert a codon-optimised HIV-1 Gag-Pol expression construct in a continuously expressed locus in 293FT cells. Subsequently Rev, envelope and vector genome expression cassettes are serially transfected. Vector titers in excess of 10 6 transducing units/ml can be harvested from the final producer clones, which can be increased to 10 8  TU/ml by concentration. This method will be of use to all basic and clinical investigators who wish to produce large batches of lentiviral vectors.

HIV-1 can incite activation of chemokine receptors, inflammatory mediators, and glutamate receptor-mediated excitotoxicity. The mechanisms associated with such immune activation can disrupt neuronal and glial functions. HIV-associated neurocognitive disorder (HAND) is being observed since the beginning of the AIDS epidemic due to a change in the functional integrity of cells from the central nervous system (CNS). Even with the presence of antiretroviral therapy, there is a decline in the functioning of the brain especially movement skills, noticeable swings in mood, and routine performance activities. Under the umbrella of HAND, various symptomatic and asymptomatic conditions are categorized and are on a rise despite the use of newer antiretroviral agents. Due to the use of long-lasting antiretroviral agents, this deadly disease is becoming a manageable chronic condition with the occurrence of asymptomatic neurocognitive impairment (ANI), symptomatic mild neurocognitive disorder, or HIV-associated dementia. In-depth research in the pathogenesis of HIV has focused on various mechanisms involved in neuronal dysfunction and associated toxicities ultimately showcasing the involvement of various pathways. Increasing evidence-based studies have emphasized a need to focus and explore the specific pathways in inflammation-associated neurodegenerative disorders. In the current review, we have highlighted the association of various HIV proteins and neuronal cells with their involvement in various pathways responsible for the development of neurotoxicity.

The HIV-1 Rev-RRE regulatory axis plays a crucial role in viral replication by facilitating the nucleo-cytoplasmic export and expression of viral mRNAs with retained introns. In this study, we investigated the impact of variation in Rev-RRE functional activity on HIV-1 replication kinetics and reactivation from latency. Using a novel HIV-1 viral vector with an interchangeable Rev cassette, we engineered viruses with two diverse Rev functional activities and demonstrated that higher Rev-RRE activity confers greater viral replication capacity while maintaining a constant level of Nef expression. In addition, a low Rev activity virus rapidly acquired a compensatory mutation in the RRE that significantly increased Rev-RRE activity and replication. In a latency model, proviruses with differing Rev-RRE activity levels varied in the efficiency of viral reactivation, affecting both initial viral release and subsequent replication kinetics. These results demonstrate that activity differences in the Rev-RRE axis among different viral isolates have important implications for HIV replication dynamics and persistence. Importantly, our findings indicate that bolstering Rev/RRE activity could be explored as part of latency reversal strategies in HIV cure efforts.

HIV-1 latency is mainly characterized at transcriptional level, and little is known about post-transcriptional mechanisms and their contribution to reactivation. The viral protein Rev controls the nucleocytoplasmic export of unspliced and singly-spliced RNA that is central to proviral replication-competence and is therefore a prerequisite for efficient viral reactivation during the “shock-and-kill” cure therapy. Here we show that during infection and reactivation, unspliced HIV-1 RNA is a subject to complex and dynamic regulation by the Rev cofactor MATR3 and the MTR4 cofactor of the nuclear exosome. MATR3 and MTR4 coexist in the same ribonucleoprotein complex functioning to either maintain or degrade the RNA, respectively, with Rev orchestrating this regulatory switch. Moreover, we provide evidence of nuclear retention of unspliced HIV-1 RNA in ex vivo cultures from 22 ART-treated people with HIV, highlighting a reversible post-transcriptional block to viral RNA nucleocytoplasmic export that is relevant to the design of curative interventions. In their study, Dorman and Bendoumou et al., reveal a post-transcriptional regulation of unspliced HIV-1 RNA by host factors MATR3, MTR4, and the viral protein Rev, identifying a previously uncharacterized post-transcriptional block in nucleocytoplasmic export, which plays a crucial role in HIV-1 latency and reactivation.

The transcription of the HIV-1 provirus results in only one type of transcript—full length genomic RNA. To make the mRNA transcripts for the accessory proteins Tat and Rev, the genomic RNA must completely splice. The mRNA transcripts for Vif, Vpr, and Env must undergo splicing but not completely. Genomic RNA (which also functions as mRNA for the Gag and Gag/Pro/Pol precursor polyproteins) must not splice at all. HIV-1 can tolerate a surprising range in the relative abundance of individual transcript types, and a surprising amount of aberrant and even odd splicing; however, it must not over-splice, which results in the loss of full-length genomic RNA and has a dramatic fitness cost. Cells typically do not tolerate unspliced/incompletely spliced transcripts, so HIV-1 must circumvent this cell policing mechanism to allow some splicing while suppressing most. Splicing is controlled by RNA secondary structure, cis-acting regulatory sequences which bind splicing factors, and the viral protein Rev. There is still much work to be done to clarify the combinatorial effects of these splicing regulators. These control mechanisms represent attractive targets to induce over-splicing as an antiviral strategy. Finally, splicing has been implicated in latency, but to date there is little supporting evidence for such a mechanism. In this review we apply what is known of cellular splicing to understand splicing in HIV-1, and present data from our newer and more sensitive deep sequencing assays quantifying the different HIV-1 transcript types.

HIV proteins target host hub proteins for transient binding interactions. The presence of viral proteins in the infected cell results in out-competition of host proteins in their interaction with hub proteins, drastically affecting cell physiology. Functional genomics and interactome datasets can be used to quantify the sequence hotspots on the HIV proteome mediating interactions with host hub proteins. In this study, we used the HIV and human interactome databases to identify HIV targeted host hub proteins and their host binding partners (H2). We developed a high throughput computational procedure utilizing motif discovery algorithms on sets of protein sequences, including sequences of HIV and H2 proteins. We identified as HIV sequence hotspots those linear motifs that are highly conserved on HIV sequences and at the same time have a statistically enriched presence on the sequences of H2 proteins. The HIV protein motifs discovered in this study are expressed by subsets of H2 host proteins potentially outcompeted by HIV proteins. A large subset of these motifs is involved in cleavage, nuclear localization, phosphorylation, and transcription factor binding events. Many such motifs are clustered on an HIV sequence in the form of hotspots. The sequential positions of these hotspots are consistent with the curated literature on phenotype altering residue mutations, as well as with existing binding site data. The hotspot map produced in this study is the first global portrayal of HIV motifs involved in altering the host protein network at highly connected hub nodes.

Nuclear export complexes composed of rev response element (RRE) ribonucleic acid (RNA) and multiple molecules of rev protein are promising targets for the development of therapeutic strategies against human immunodeficiency virus type 1 (HIV-1), but their assembly remains poorly understood. Using native mass spectrometry, we show here that rev initially binds to the upper stem of RRE IIB, from where it is relayed to binding sites that allow for rev dimerization. The newly discovered binding region implies initial rev recognition by nucleotides that are not part of the internal loop of RRE stem IIB RNA, which was previously identified as the preferred binding region. Our study highlights the unique capability of native mass spectrometry to separately study the binding interfaces of RNA/protein complexes of different stoichiometry, and provides a detailed understanding of the mechanism of RRE/rev association with implications for the rational design of potential drugs against HIV-1 infection. The HIV-1 RNA-binding protein rev facilitates nuclear export of viral RNA. Here, the authors use native mass spectrometry to study the interactions between rev-derived peptides and rev response elements of HIV-1 RNA, providing mechanistic insights into rev recognition and recruitment.

HIV-1 Rev is a small regulatory protein that mediates the nuclear export of viral mRNAs, an essential step in the HIV replication cycle. In this process Rev oligomerizes in association with a highly structured RNA motif, the Rev response element. Crystallographic studies of Rev have been hampered by the protein's tendency to aggregate, but Rev has now been found to form a stable soluble equimolar complex with a specifically engineered monoclonal Fab fragment. We have determined the structure of this complex at 3.2 Å resolution. It reveals a molecular dimer of Rev, bound on either side by a Fab, where the ordered portion of each Rev monomer (residues 9-65) contains two coplanar α-helices arranged in hairpin fashion. Subunits dimerize through overlapping of the hairpin prongs. Mating of hydrophobic patches on the outer surface of the dimer is likely to promote higher order interactions, suggesting a model for Rev oligomerization onto the viral RNA.

Nuclear HIV-1 mRNA export is mediated by cooperative Rev protein binding to the Rev response element (RRE) RNA, forming a complex recognized by the Crm1 host export factor. A structure of a Rev dimer now shows that the organization of Rev monomers within a dimer defines the RRE recognition interface, with the other side likely binding Crm1. HIV replication requires nuclear export of unspliced viral RNAs to translate structural proteins and package genomic RNA. Export is mediated by cooperative binding of the Rev protein to the Rev response element (RRE) RNA, to form a highly specific oligomeric ribonucleoprotein (RNP) that binds to the Crm1 host export factor. To understand how protein oligomerization generates cooperativity and specificity for RRE binding, we solved the crystal structure of a Rev dimer at 2.5-Å resolution. The dimer arrangement organizes arginine-rich helices at the ends of a V-shaped assembly to bind adjacent RNA sites and structurally couple dimerization and RNA recognition. A second protein-protein interface arranges higher-order Rev oligomers to act as an adaptor to the host export machinery, with viral RNA bound to one face and Crm1 to another, the oligomers thereby using small, interconnected modules to physically arrange the RNP for efficient export.