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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.

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.

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.

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-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.

HIV-1-infected people who take drugs that suppress viremia to undetectable levels are protected from developing AIDS. Nonetheless, HIV-1 establishes proviruses in long-lived CD4 + memory T cells, and perhaps other cell types, that preclude elimination of the virus even after years of continuous antiviral therapy. Here we show that the HIV-1 provirus activates innate immune signaling in isolated dendritic cells, macrophages, and CD4 + T cells. Immune activation requires transcription from the HIV-1 provirus and expression of CRM1-dependent, Rev-dependent, RRE-containing, unspliced HIV-1 RNA. If rev is provided in trans , all HIV-1 coding sequences are dispensable for activation except those cis -acting sequences required for replication or splicing. Our results indicate that the complex, post-transcriptional regulation intrinsic to HIV-1 RNA is detected by the innate immune system as a danger signal, and that drugs which disrupt HIV-1 transcription or HIV-1 RNA metabolism would add qualitative benefit to current antiviral drug regimens. During HIV infection, antiviral therapy can suppress viraemia to undetectable levels and hinder the progression towards AIDS; however the HIV-1 provirus can remain in long-lived CD4 +  memory T cells. Here the authors show that intronic RNA from the HIV-1 provirus can induce type I interferon and inflammatory cytokine production.

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.

The ability of the retroviral Gag protein of Rous sarcoma virus (RSV) to transiently traffic through the nucleus is well-established and has been implicated in genomic RNA (gRNA) packaging Although other retroviral Gag proteins (human immunodeficiency virus type 1, HIV-1; feline immunodeficiency virus, FIV; Mason-Pfizer monkey virus, MPMV; mouse mammary tumor virus, MMTV; murine leukemia virus, MLV; and prototype foamy virus, PFV) have also been observed in the nucleus, little is known about what, if any, role nuclear trafficking plays in those viruses. In the case of HIV-1, the Gag protein interacts in nucleoli with the regulatory protein Rev, which facilitates nuclear export of gRNA. Based on the knowledge that RSV Gag forms viral ribonucleoprotein (RNPs) complexes with unspliced viral RNA (USvRNA) in the nucleus, we hypothesized that the interaction of HIV-1 Gag with Rev could be mediated through vRNA to form HIV-1 RNPs. Using inducible HIV-1 proviral constructs, we visualized HIV-1 Gag and USvRNA in discrete foci in the nuclei of HeLa cells by confocal microscopy. Two-dimensional co-localization and RNA-immunoprecipitation of fractionated cells revealed that interaction of nuclear HIV-1 Gag with USvRNA was specific. Interestingly, treatment of cells with transcription inhibitors reduced the number of HIV-1 Gag and USvRNA nuclear foci, yet resulted in an increase in the degree of Gag co-localization with USvRNA, suggesting that Gag accumulates on newly synthesized viral transcripts. Three-dimensional imaging analysis revealed that HIV-1 Gag localized to the perichromatin space and associated with USvRNA and Rev in a tripartite RNP complex. To examine a more biologically relevant cell, latently infected CD4+ T cells were treated with prostratin to stimulate NF-κB mediated transcription, demonstrating striking localization of full-length Gag at HIV-1 transcriptional burst site, which was labelled with USvRNA-specific riboprobes. In addition, smaller HIV-1 RNPs were observed in the nuclei of these cells. These data suggest that HIV-1 Gag binds to unspliced viral transcripts produced at the proviral integration site, forming vRNPs in the nucleus.

During HIV infection, intron-containing viral mRNAs are exported from the cell nucleus to the cytoplasm to complete the replication cycle. Cellular restrictions on the export of incompletely spliced transcripts are overcome by a viral protein, Rev, and an RNA structure found in all unspliced and incompletely spliced viral mRNAs, the Rev Response Element (RRE). Primary HIV isolates display substantial variation in the sequence and functional activity of Rev proteins. We analyzed Rev from two primary isolates with disparate activity that resulted in differences in in vitro fitness of replication-competent viral constructs. The results showed that amino acid differences within the oligomerization domain, but not the arginine-rich motif or the nuclear export signal, determined the level of Rev activity. Two specific amino acid substitutions were sufficient to alter the low-activity Rev to a high-activity phenotype. Other mutations in Rev sequences had unpredictable effects on activity that differed between the two Rev backbones. The sensitivity of Rev function level to small sequence changes likely permits modulation of Rev-RRE activity during HIV infection, which may play a role in pathogenesis. The functional consequences of Rev mutations differed between primary isolates, highlighting the challenge of generalizing studies of Rev conducted using laboratory HIV strains.

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.