Catalytically inactive dCas9 fused to transcriptional activators (dCas9-VPR) enables activation of silent genes. Many disease genes have counterparts, which serve similar functions but are expressed in distinct cell types. One attractive option to compensate for the missing function of a defective gene could be to transcriptionally activate its functionally equivalent counterpart via dCas9-VPR. Key challenges of this approach include the delivery of dCas9-VPR, activation efficiency, long-term expression of the target gene, and adverse effects in vivo. Using dual adeno-associated viral vectors expressing split dCas9-VPR, we show efficient transcriptional activation and long-term expression of cone photoreceptor-specific M-opsin (Opn1mw) in a rhodopsin-deficient mouse model for retinitis pigmentosa. One year after treatment, this approach yields improved retinal function and attenuated retinal degeneration with no apparent adverse effects. Our study demonstrates that dCas9-VPR-mediated transcriptional activation of functionally equivalent genes has great potential for the treatment of genetic disorders.

Numerous studies have reported that Vpr alters NF-κB signaling in various cell types, however, the findings have been largely conflicting with reports of both stimulatory and inhibitory effects of Vpr. Our aim was to investigate the role of Vpr signaling in myeloid cells using an adenovirus based expression and indicator system. Our results show that Vpr is inhibitory to NF-κB, however, this effect is dependent on the particular manner of NF-κB stimulation. Consistent with this notion, we report that Vpr has inhibitory effects that are specific to the TNF-α pathway, but not affecting the LPS pathway, suggesting that differential targets of Vpr may exist for NF-κB regulation. Further, we identify VprBP as one possible cellular component of Vpr's regulation of IκBα in response to TNF-α stimulation. We did not identify such a role for HSP27, which instead seems to inhibit Vpr functions. Chronically HIV-1 infected U1 cells with knockdown constructs for Vpr were unexpectedly less responsive to TNF-α mediated viral replication, perhaps suggesting that other HIV-1 components may antagonize these anti-NF-κB effects in infected cells. We hypothesize that Vpr may serve an important role in the context of viral infection and immune function in vivo, through its selective inhibition of NF-κB pathways.

The HIV-1 Vpr accessory protein induces ubiquitin/proteasome-dependent degradation of many cellular proteins by recruiting them to a cullin4A-DDB1-DCAF1 complex. In so doing, Vpr enhances HIV-1 gene expression and induces (G2/M) cell cycle arrest. However, the identities of Vpr target proteins through which these biological effects are exerted are unknown. We show that a chromosome periphery protein, CCDC137/cPERP-B, is targeted for depletion by HIV-1 Vpr, in a cullin4A-DDB1-DCAF1 dependent manner. CCDC137 depletion caused G2/M cellcycle arrest, while Vpr-resistant CCDC137 mutants conferred resistance to Vpr-induced G2/M arrest. CCDC137 depletion also recapitulated the ability of Vpr to enhance HIV-1 gene expression, particularly in macrophages. Our findings indicate that Vpr promotes cell-cycle arrest and HIV-1 gene expression through depletion of CCDC137.

HIV-1 Vpr is necessary for maximal HIV infection and spread in macrophages. Evolutionary conservation of Vpr suggests an important yet poorly understood role for macrophages in HIV pathogenesis. Vpr counteracts a previously unknown macrophage-specific restriction factor that targets and reduces the expression of HIV Env. Here, we report that the macrophage mannose receptor (MR), is a restriction factor targeting Env in primary human monocyte-derived macrophages. Vpr acts synergistically with HIV Nef to target distinct stages of the MR biosynthetic pathway and dramatically reduce MR expression. Silencing MR or deleting mannose residues on Env rescues Env expression in HIV-1-infected macrophages lacking Vpr. However, we also show that disrupting interactions between Env and MR reduces initial infection of macrophages by cell-free virus. Together these results reveal a Vpr-Nef-Env axis that hijacks a host mannose-MR response system to facilitate infection while evading MR's normal role, which is to trap and destroy mannose-expressing pathogens.

The HIV-1 accessory proteins Vif, Vpu, and Nef can promote infection by overcoming the inhibitory effects of the host cell restriction factors APOBEC3G, Tetherin, and SERINC5, respectively. However, how the HIV-1 accessory protein Vpr enhances infection in macrophages but not in CD4+ T cells remains elusive. Here, we report that Vpr counteracts lysosomal-associated transmembrane protein 5 (LAPTM5), a potent inhibitor of HIV-1 particle infectivity, to enhance HIV-1 infection in macrophages. LAPTM5 transports HIV-1 envelope glycoproteins to lysosomes for degradation, thereby inhibiting virion infectivity. Vpr counteracts the restrictive effects of LAPTM5 by triggering its degradation via DCAF1. In the absence of Vpr, the silencing of LAPTM5 precisely phenocopied the effect of Vpr on HIV-1 infection. In contrast, Vpr did not enhance HIV-1 infection in the absence of LAPTM5. Moreover, LAPTM5 was highly expressed in macrophages but not in CD4+ T lymphocytes. Re-expressing LAPTM5 reconstituted the Vpr-dependent promotion of HIV-1 infection in primary CD4+ T cells, as observed in macrophages. Herein, we demonstrate the molecular mechanism used by Vpr to overcome LAPTM5 restriction in macrophages, providing a potential strategy for anti-HIV/AIDS therapeutics.

The HIV-1 accessory protein, Vpr, is an enigmatic protein required for efficient spread of HIV from macrophages to T cells, a necessary step for propagation of infection. To illuminate the role of Vpr in HIV-infection of primary macrophages, we used single-cell RNA sequencing to capture the transcriptional changes during an HIV-1 spreading infection plus and minus Vpr. We found that Vpr reprogramed HIV-infected macrophage gene expression by targeting the master transcriptional regulator, PU.1. PU.1 was required for efficient induction of the host innate immune response to HIV, including upregulation of ISG15 , LY96, and IFI6 . In contrast, we did not observe direct effects of PU.1 on HIV gene transcription. Single cell gene expression analysis also revealed Vpr countered an innate immune response to HIV-infection within bystander macrophages via a PU.1-independent mechanism. The capacity of Vpr to target PU.1 and disrupt the anti-viral response was highly conserved across primate lentiviruses including HIV-2 and several SIVs. By demonstrating how Vpr overcomes a critical early warning system of infection, we identify a crucial reason why Vpr is necessary for HIV infection and spread.

Viruses of human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency virus (SIV) lineages carry a unique accessory protein called Vpx. Vpx is essential or critical for viral replication in natural target cells such as macrophages and T lymphocytes. We have previously shown that a poly-proline motif (PPM) located at the C-terminal region of Vpx is required for its efficient expression in two strains of HIV-2 and SIVmac, and that the Vpx expression levels of the two clones are significantly different. Notably, the PPM sequence is conserved and confined to Vpx and Vpr proteins derived from certain lineages of HIV-2/SIVs. In this study, Vpx/Vpr proteins from diverse primate lentiviral lineages were experimentally and phylogenetically analyzed to obtain the general expression picture in cells. While both the level and PPM-dependency of Vpx/Vpr expression in transfected cells varied among viral strains, each viral group, based on Vpx/Vpr amino acid sequences, was found to exhibit a characteristic expression profile. Moreover, phylogenetic tree analyses on Gag and Vpx/Vpr proteins gave essentially the same results. Taken together, our study described here suggests that each primate lentiviral lineage may have developed a unique expression pattern of Vpx/Vpr proteins for adaptation to its hostile cellular and species environments in the process of viral evolution.

Lentiviruses, including HIV-1, possess the ability to enter the nucleus through nuclear pore complexes and can infect interphase cells, including those actively replicating chromosomal DNA. Viral accessory proteins hijack host cell E3 enzymes to antagonize intrinsic defenses, and thereby provide a more permissive environment for virus replication. The HIV-1 Vpr accessory protein reprograms CRL4DCAF1 E3 to antagonize select postreplication DNA repair enzymes and activates the DNA damage checkpoint in the G2 cell cycle phase. However, little is known about the roles played by these Vpr targets in HIV-1 replication. Here, using a sensitive pairwise replication competition assay, we show that Vpr endows HIV-1 with a strong replication advantage in activated primary CD4+ T cells and established T cell lines. This effect is disabled by a Vpr mutation that abolishes binding to CRL4DCAF1 E3, thereby disrupting Vpr antagonism of helicase-like transcription factor (HLTF) DNA helicase and other DNA repair pathway targets, and by another mutation that prevents induction of the G2 DNA damage checkpoint. Consistent with these findings, we also show that HLTF restricts HIV-1 replication, and that this restriction is antagonized by HIV-1 Vpr. Furthermore, our data imply that HIV-1 Vpr uses additional, yet to be identified mechanisms to facilitate HIV-1 replication in T cells. Overall, we demonstrate that multiple aspects of the cellular DNA repair machinery restrict HIV-1 replication in dividing T cells, the primary target of HIV-1 infection, and describe newly developed approaches to dissect key components.

The enormous genetic variability reported in HIV-1 has posed problems in the treatment of infected individuals. This is evident in the form of HIV-1 resistant to antiviral agents, neutralizing antibodies and cytotoxic T lymphocytes (CTLs) involving multiple viral gene products. Based on this, it has been suggested that a comprehensive analysis of the polymorphisms in HIV proteins is of value for understanding the virus transmission and pathogenesis as well as for the efforts towards developing anti-viral therapeutics and vaccines. This study, for the first time, describes an in-depth analysis of genetic variation in Vpr using information from global HIV-1 isolates involving a total of 976 Vpr sequences. The polymorphisms at the individual amino acid level were analyzed. The residues 9, 33, 39, and 47 showed a single variant amino acid compared to other residues. There are several amino acids which are highly polymorphic. The residues that show ten or more variant amino acids are 15, 16, 28, 36, 37, 48, 55, 58, 59, 77, 84, 86, 89, and 93. Further, the variant amino acids noted at residues 60, 61, 34, 71 and 72 are identical. Interestingly, the frequency of the variant amino acids was found to be low for most residues. Vpr is known to contain multiple CTL epitopes like protease, reverse transcriptase, Env, and Gag proteins of HIV-1. Based on this, we have also extended our analysis of the amino acid polymorphisms to the experimentally defined and predicted CTL epitopes. The results suggest that amino acid polymorphisms may contribute to the immune escape of the virus. The available data on naturally occurring polymorphisms will be useful to assess their potential effect on the structural and functional constraints of Vpr and also on the fitness of HIV-1 for replication.

Previously, we reported that cellular transcription factor ZASC1 facilitates DNA-dependent/RNA-independent recruitment of HIV-1 TAT and the cellular elongation factor P-TEFb to the HIV-1 promoter and is a critical factor in regulating HIV-1 transcriptional elongation (PLoS Path e1003712). Here we report that cellular transcription factor ZBTB2 is a novel repressor of HIV-1 gene expression. ZBTB2 strongly co-immunoprecipitated with ZASC1 and was dramatically relocalized by ZASC1 from the cytoplasm to the nucleus. Mutations abolishing ZASC1/ZBTB2 interaction prevented ZBTB2 nuclear relocalization. We show that ZBTB2-induced repression depends on interaction of cellular histone deacetylases (HDACs) with the ZBTB2 POZ domain. Further, ZASC1 interaction specifically recruited ZBTB2 to the HIV-1 promoter, resulting in histone deacetylation and transcription repression. Depleting ZBTB2 by siRNA knockdown or CRISPR/CAS9 knockout in T cell lines enhanced transcription from HIV-1 vectors lacking Vpr, but not from these vectors expressing Vpr. Since HIV-1 Vpr activates the viral LTR by inducing the ATR kinase/DNA damage response pathway, we investigated ZBTB2 response to Vpr and DNA damaging agents. Expressing Vpr or stimulating the ATR pathway with DNA damaging agents impaired ZASC1's ability to localize ZBTB2 to the nucleus. Moreover, the effects of DNA damaging agents and Vpr on ZBTB2 localization could be blocked by ATR kinase inhibitors. Critically, Vpr and DNA damaging agents decreased ZBTB2 binding to the HIV-1 promoter and increased promoter histone acetylation. Thus, ZBTB2 is recruited to the HIV-1 promoter by ZASC1 and represses transcription, but ATR pathway activation leads to ZBTB2 removal from the promoter, cytoplasmic sequestration and activation of viral transcription. Together, our data show that ZASC1/ZBTB2 integrate the functions of TAT and Vpr to maximize HIV-1 gene expression.

The Vpr protein of human immunodeficiency virus type 1 (HIV-1) contributes to viral replication in non-dividing cells, specifically those of the myeloid lineage. However, the effects of Vpr in enhancing HIV-1 infection in dendritic cells have not been extensively investigated. Here, we evaluated the role of Vpr during infection of highly permissive peripheral blood mononuclear cells (PBMCs) and CD4(+) T-cells and compared it to that of monocyte-derived dendritic cells (MDDCs), which are less susceptible to HIV-1 infection. Infections of dividing PBMCs and non-dividing MDDCs were carried out with single-cycle and replication-competent HIV-1 encoding intact Vpr or Vpr-defective mutants. In contrast to previous findings, we observed that single-cycle HIV-1 infection of both PBMCs and MDDCs was significantly enhanced in the presence of Vpr when the viral stocks were carefully characterized and titrated. HIV-1 DNA quantification revealed that Vpr only enhanced the reverse transcription and nuclear import processes in single-cycle HIV-1 infected MDDCs, but not in CD4(+) T-cells. However, a significant enhancement in HIV-1 gag mRNA expression was observed in both CD4(+) T-cells and MDDCs in the presence of Vpr. Furthermore, Vpr complementation into HIV-1 virions did not affect single-cycle viral infection of MDDCs, suggesting that newly synthesized Vpr plays a significant role to facilitate single-cycle HIV-1 infection. Over the course of a spreading infection, Vpr significantly enhanced replication-competent HIV-1 infection in MDDCs, while it modestly promoted viral infection in activated PBMCs. Quantification of viral DNA in replication-competent HIV-1 infected PBMCs and MDDCs revealed similar levels of reverse transcription products, but increased nuclear import in the presence of Vpr independent of the cell types. Taken together, our results suggest that Vpr has differential effects on single-cycle and spreading HIV-1 infections, which are dependent on the permissiveness of the target cell.

HIV-1 replicates in CD4+ T cells, leading to AIDS. Determining how HIV-1 shapes its niche to create a permissive environment is central to informing efforts to limit pathogenesis, disturb reservoirs, and achieve a cure. A key roadblock in understanding HIV-T cell interactions is the requirement to activate T cells in vitro to make them permissive to infection. This dramatically alters T cell biology and virus-host interactions. Here we show that HIV-1 cell-to-cell spread permits efficient, productive infection of resting memory T cells without prior activation. Strikingly, we find that HIV-1 infection primes resting T cells to gain characteristics of tissue-resident memory T cells (TRM), including upregulating key surface markers and the transcription factor Blimp-1 and inducing a transcriptional program overlapping the core TRM transcriptional signature. This reprogramming is driven by Vpr and requires Vpr packaging into virions and manipulation of STAT5. Thus, HIV-1 reprograms resting T cells, with implications for viral replication and persistence.

Transgenic animals with increased or abrogated target gene expression are powerful tools for drug discovery research. Here, we developed a CRISPR-based Rosa26-LSL-dCas9-VPR mouse model for targeted induction of endogenous gene expression using different Adeno-associated virus (AAV) capsid variants for tissue-specific gRNAs delivery. To show applicability of the model, we targeted low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9), either individually or together. We induced up to ninefold higher expression of hepatocellular proteins. In consequence of LDLR upregulation, plasma LDL levels almost abolished, whereas upregulation of PCSK9 led to increased plasma LDL and cholesterol levels. Strikingly, simultaneous upregulation of both LDLR and PCSK9 resulted in almost unaltered LDL levels. Additionally, we used our model to achieve expression of all α1-Antitrypsin (AAT) gene paralogues simultaneously. These results show the potential of our model as a versatile tool for optimized targeted gene expression, alone or in combination.

Integration site landscapes, clonal dynamics, and latency reversal with or without vpr were compared in HIV-1-infected Jurkat cell populations, and the properties of individual clones were defined. Clones differed in fractions of long terminal repeat (LTR)-active daughter cells, with some clones containing few to no LTR-active cells, while almost all cells were LTR active for others. Clones varied over 4 orders of magnitude in virus release per active cell. Proviruses in largely LTR-active clones were closer to preexisting enhancers and promoters than low-LTR-active clones. Unsurprisingly, major vpr+ clones contained fewer LTR-active cells than vpr- clones, and predominant vpr+ proviruses were farther from enhancers and promoters than those in vpr- pools. Distances to these marks among intact proviruses previously reported for antiretroviral therapy (ART)-suppressed patients revealed that patient integration sites were more similar to those in the vpr+ pool than to vpr- integrants. Complementing vpr-defective proviruses with vpr led to the rapid loss of highly LTR-active clones, indicating that the effect of Vpr on proviral populations occurred after integration. However, major clones in the complemented pool and its vpr- parent population did not differ in burst sizes. When the latency reactivation agents prostratin and JQ1 were applied separately or in combination, vpr+ and vpr- population-wide trends were similar, with dual-treatment enhancement being due in part to reactivated clones that did not respond to either drug applied separately. However, the expression signatures of individual clones differed between populations. These observations highlight how Vpr, exerting selective pressure on proviral epigenetic variation, can shape integration site landscapes, proviral expression patterns, and reactivation properties. IMPORTANCE A bedrock assumption in HIV-1 population modeling is that all active cells release the same amount of virus. However, the findings here revealed that when HIV-infected cells expand into clones, each clone differs in virus production. Reasoning that this variation in expression patterns constituted a population of clones from which differing subsets would prevail under differing environmental conditions, the cytotoxic HIV-1 protein Vpr was introduced, and population dynamics and expression properties were compared in the presence and absence of Vpr. The results showed that whereas most clones produced fairly continuous levels of virus in the absence of Vpr, its presence selected for a distinct subset of clones with properties reminiscent of persistent populations in patients, suggesting the possibility that the interclonal variation in expression patterns observed in culture may contribute to proviral persistence in vivo.

Although apoptosis is considered one of the major mechanisms of CD4(+) T cell depletion in HIV-infected patients, the virus-infected cells somehow appear to be protected from apoptosis, which generally occurs in bystander cells. Vpr is an auxiliary HIV-1 protein, which, unlike the other regulatory gene products, is present at high copy number in virus particles. We established stable transfectants of CD4+ T Jurkat cells constitutively expressing low levels of vpr. These clones exhibited cell cycle characteristics similar to those of control-transfected cells. Treatment of control clones with apoptotic stimuli (i.e., cycloheximide/tumor necrosis factor alpha (TNF-alpha), anti-Fas antibody, or serum starvation) resulted in a massive cell death by apoptosis. In contrast, all the vpr-expressing clones showed an impressive protection from apoptosis independently of the inducer. Notably, vpr antisense phosphorothioate oligodeoxynucleotides render vpr-expressing cells as susceptible to apoptosis induced by cycloheximide and TNF-alpha as the control clones. Moreover, the constitutive expression of HIV-1 vpr resulted in the upregulation of bcl-2, an oncogene endowed with antiapoptotic activities, and in the downmodulation of bax, a proapoptotic factor of the bcl-2 family. Altogether, these results suggest that low levels of the endogenous vpr protein can interfere with the physiological turnover of T lymphocytes at early stages of virus infection, thus facilitating HIV persistence and, subsequently, viral spread. This might explain why apoptosis mostly occurs in bystander uninfected cells in AIDS patients.

Forkhead box P3+ (FOXP3 +) regulatory T cells (Tregs) are a subset of lymphocytes, critical for the maintenance of immune homeostasis. Loss-of-function mutations of the FOXP3 gene in animal models and humans results in loss of differentiation potential into Treg cells and are responsible for several immune-mediated inflammatory diseases. Strategies of increasing FOXP3 expression represent a potential approach to increase the pool of Tregs within the lymphocyte population and may be employed in therapies of diverse autoimmune conditions. In the present study, a dCas9 CRISPR-based method was systematically employed to achieve upregulation and sustained high expression of endogenous FOXP3 in HEK293 and human Jurkat T cell lines through targeting of the core promotor, three known regulatory regions of the FOXP3 gene (CNS1-3), and two additional regions selected through extensive bioinformatics analysis (Cage1 and Cage2).

Programmable control of gene expression via nuclease-null Cas9 fusion proteins has enabled the engineering of cellular behaviors. Here, both transcriptional and epigenetic gene activation via synthetic mRNA and lipid nanoparticle delivery was demonstrated in vivo. These highly efficient delivery strategies resulted in high levels of activation in multiple tissues. Finally, we demonstrate durable gene activation in vivo via transient delivery of a single dose of a gene activator that combines VP64, p65, and HSF1 with a SWI/SNF chromatin remodeling complex component SS18, representing an important step toward gene-activation-based therapeutics. This induced sustained gene activation could be inhibited via mRNA-encoded AcrIIA4, further improving the safety profile of this approach.

Liquid-liquid phase separation (LLPS) plays a critical role in regulating gene transcription via the formation of transcriptional condensates. However, LLPS has not been reported to be engineered as a tool to activate endogenous gene expression in mammalian cells or in vivo. Here, we developed a droplet-forming CRISPR (clustered regularly interspaced short palindromic repeats) gene activation system (DropCRISPRa) to activate transcription with high efficiency via combining the CRISPR-SunTag system with FETIDR-AD fusion proteins, which contain an N-terminal intrinsically disordered region (IDR) of a FET protein (FUS or TAF15) and a transcription activation domain (AD, VP64/P65/VPR). In this system, the FETIDR-AD fusion protein formed phase separation condensates at the target sites, which could recruit endogenous BRD4 and RNA polymerase II with an S2 phosphorylated C-terminal domain (CTD) to enhance transcription elongation. IDR-FUS9Y>S and IDR-FUSG156E, two mutants with deficient and aberrant phase separation respectively, confirmed that appropriate phase separation was required for efficient gene activation. Further, the DropCRISPRa system was compatible with a broad set of CRISPR-associated (Cas) proteins and ADs, including dLbCas12a, dAsCas12a, dSpCas9 and the miniature dUnCas12f1, and VP64, P65 and VPR. Finally, the DropCRISPRa system could activate target genes in mice. Therefore, this study provides a robust tool to activate gene expression for foundational research and potential therapeutics.

CRISPR-based technology has provided new avenues to interrogate gene function, but difficulties in transgene expression in post-mitotic neurons has delayed incorporation of these tools in the central nervous system (CNS). Here, we demonstrate a highly efficient, neuron-optimized dual lentiviral CRISPR-based transcriptional activation (CRISPRa) system capable of robust, modular, and tunable gene induction and multiplexed gene regulation across several primary rodent neuron culture systems. CRISPRa targeting unique promoters in the complex multi-transcript gene brain-derived neurotrophic factor (Bdnf) revealed both transcript- and genome-level selectivity of this approach, in addition to highlighting downstream transcriptional and physiological consequences of Bdnf regulation. Finally, we illustrate that CRISPRa is highly efficient in vivo, resulting in increased protein levels of a target gene in diverse brain structures. Taken together, these results demonstrate that CRISPRa is an efficient and selective method to study gene expression programs in brain health and disease.

CRISPR-interference (CRISPRi), a highly effective method for silencing genes in mammalian cells, employs an enzymatically dead form of Cas9 (dCas9) complexed with one or more guide RNAs (gRNAs) with 20 nucleotides (nt) of complementarity to transcription initiation sites of target genes. Such gRNA/dCas9 complexes bind to DNA, impeding transcription of the targeted locus. Here, we present an alternative gene-suppression strategy using active Cas9 complexed with truncated gRNAs (tgRNAs). Cas9/tgRNA complexes bind to specific target sites without triggering DNA cleavage. When targeted near transcriptional start sites, these short 14-15 nts tgRNAs efficiently repress expression of several target genes throughout somatic tissues in Drosophila melanogaster without generating any detectable target site mutations. tgRNAs also can activate target gene expression when complexed with a Cas9-VPR fusion protein or modulate enhancer activity, and can be incorporated into a gene-drive, wherein a traditional gRNA sustains drive while a tgRNA inhibits target gene expression.