Comprehensive synergy mapping links a BAF- and NSL-containing "supercomplex" to the transcriptional silencing of HIV-1.

Host repressors mediate HIV latency, but how they interactively silence the virus remains unclear. Here, we develop "reiterative enrichment and authentication of CRISPRi targets for synergies (REACTS)" to probe the genome for synergies between HIV transcription repressors. Using eight known host repressors as queries, we identify 32 synergies involving eleven repressors, including BCL7C, KANSL2, and SIRT2. Overexpression of these three proteins reduces HIV reactivation in Jurkat T cells and in CD4 T cells from people living with HIV on antiretroviral therapy (ART). We show that the BCL7C-containing BAF complex and the KANSL2-containing NSL complex form a "supercomplex" that increases inhibitory histone acetylation of the HIV long-terminal repeat (LTR) and its occupancy by the short variant of the acetyl-lysine reader Brd4. Collectively, we provide a validated platform for defining gene synergies genome wide, and the BAF-NSL "supercomplex" represents a potential target for overcoming HIV rebound after ART cessation.

Identification of unrecognized host factors promoting HIV-1 latency.

To counter HIV latency, it is important to develop a better understanding of the full range of host factors promoting latency. Their identification could suggest new strategies to reactivate latent proviruses and subsequently kill the host cells ("shock and kill"), or to permanently silence these latent proviruses ("block and lock"). We recently developed a screening strategy termed "Reiterative Enrichment and Authentication of CRISPRi Targets" (REACT) that can unambiguously identify host genes promoting HIV latency, even in the presence of high background "noise" produced by the stochastic nature of HIV reactivation. After applying this strategy in four cell lines displaying different levels of HIV inducibility, we identified FTSJ3, TMEM178A, NICN1 and the Integrator Complex as host genes promoting HIV latency. shRNA knockdown of these four repressive factors significantly enhances HIV expression in primary CD4 T cells, and active HIV infection is preferentially found in cells expressing lower levels of these four factors. Mechanistically, we found that downregulation of these newly identified host inhibitors stimulates different stages of RNA Polymerase II-mediated transcription of HIV-1. The identification and validation of these new host inhibitors provide insight into the novel mechanisms that maintain HIV latency even when cells are activated and undergo cell division.

Reiterative Enrichment and Authentication of CRISPRi Targets (REACT) identifies the proteasome as a key contributor to HIV-1 latency.

The establishment of HIV-1 latency gives rise to persistent chronic infection that requires life-long treatment. To reverse latency for viral eradiation, the HIV-1 Tat protein and its associated ELL2-containing Super Elongation Complexes (ELL2-SECs) are essential to activate HIV-1 transcription. Despite efforts to identify effective latency-reversing agents (LRA), avenues for exposing latent HIV-1 remain inadequate, prompting the need to identify novel LRA targets. Here, by conducting a CRISPR interference-based screen to reiteratively enrich loss-of-function genotypes that increase HIV-1 transcription in latently infected CD4+ T cells, we have discovered a key role of the proteasome in maintaining viral latency. Downregulating or inhibiting the proteasome promotes Tat-transactivation in cell line models. Furthermore, the FDA-approved proteasome inhibitors bortezomib and carfilzomib strongly synergize with existing LRAs to reactivate HIV-1 in CD4+ T cells from antiretroviral therapy-suppressed individuals without inducing cell activation or proliferation. Mechanistically, downregulating/inhibiting the proteasome elevates the levels of ELL2 and ELL2-SECs to enable Tat-transactivation, indicating the proteasome-ELL2 axis as a key regulator of HIV-1 latency and promising target for therapeutic intervention.

The KAT5-Acetyl-Histone4-Brd4 axis silences HIV-1 transcription and promotes viral latency.

The bromodomain protein Brd4 promotes HIV-1 latency by competitively inhibiting P-TEFb-mediated transcription induced by the virus-encoded Tat protein. Brd4 is recruited to the HIV LTR by interactions with acetyl-histones3 (AcH3) and AcH4. However, the precise modification pattern that it reads and the writer for generating this pattern are unknown. By examining a pool of latently infected proviruses with diverse integration sites, we found that the LTR characteristically has low AcH3 but high AcH4 content. This unusual acetylation profile attracts Brd4 to suppress the interaction of Tat with the host super elongation complex (SEC) that is essential for productive HIV transcription and latency reversal. KAT5 (lysine acetyltransferase 5), but not its paralogs KAT7 and KAT8, is found to promote HIV latency through acetylating H4 on the provirus. Antagonizing KAT5 removes AcH4 and Brd4 from the LTR, enhances the SEC loading, and reverses as well as delays, the establishment of latency. The pro-latency effect of KAT5 is confirmed in a primary CD4+ T cell latency model as well as cells from ART-treated patients. Our data thus indicate the KAT5-AcH4-Brd4 axis as a key regulator of latency and a potential therapeutic target to reactivate latent HIV reservoirs for eradication.

Structural basis for ELL2 and AFF4 activation of HIV-1 proviral transcription.

The intrinsically disordered scaffold proteins AFF1/4 and the transcription elongation factors ELL1/2 are core components of the super elongation complex required for HIV-1 proviral transcription. Here we report the 2.0-Å resolution crystal structure of the human ELL2 C-terminal domain bound to its 50-residue binding site on AFF4, the ELLBow. The ELL2 domain has the same arch-shaped fold as the tight junction protein occludin. The ELLBow consists of an N-terminal helix followed by an extended hairpin that we refer to as the elbow joint, and occupies most of the concave surface of ELL2. This surface is important for the ability of ELL2 to promote HIV-1 Tat-mediated proviral transcription. The AFF4-ELL2 interface is imperfectly packed, leaving a cavity suggestive of a potential binding site for transcription-promoting small molecules.

A Minor Subset of Super Elongation Complexes Plays a Predominant Role in Reversing HIV-1 Latency.

Promoter-proximal pausing by RNA polymerase II (Pol II) is a key rate-limiting step in HIV-1 transcription and latency reversal. The viral Tat protein recruits human super elongation complexes (SECs) to paused Pol II to overcome this restriction. Despite the recent progress in understanding the functions of different subsets of SECs in controlling cellular and Tat-activated HIV transcription, little is known about the SEC subtypes that help reverse viral latency in CD4(+) T cells. Here, we used the CRISPR-Cas9 genome-editing tool to knock out the gene encoding the SEC subunit ELL2, AFF1, or AFF4 in Jurkat/2D10 cells, a well-characterized HIV-1 latency model. Depletion of these proteins drastically reduced spontaneous and drug-induced latency reversal by suppressing HIV-1 transcriptional elongation. Surprisingly, a low-abundance subset of SECs containing ELL2 and AFF1 was found to play a predominant role in cooperating with Tat to reverse latency. By increasing the cellular level/activity of these Tat-friendly SECs, we could potently activate latent HIV-1 without using any drugs. These results implicate the ELL2/AFF1-SECs as an important target in the future design of a combinatorial therapeutic approach to purge latent HIV-1.

Gene target specificity of the Super Elongation Complex (SEC) family: how HIV-1 Tat employs selected SEC members to activate viral transcription.

The AF4/FMR2 proteins AFF1 and AFF4 act as a scaffold to assemble the Super Elongation Complex (SEC) that strongly activates transcriptional elongation of HIV-1 and cellular genes. Although they can dimerize, it is unclear whether the dimers exist and function within a SEC in vivo. Furthermore, it is unknown whether AFF1 and AFF4 function similarly in mediating SEC-dependent activation of diverse genes. Providing answers to these questions, our current study shows that AFF1 and AFF4 reside in separate SECs that display largely distinct gene target specificities. While the AFF1-SEC is more potent in supporting HIV-1 transactivation by the viral Tat protein, the AFF4-SEC is more important for HSP70 induction upon heat shock. The functional difference between AFF1 and AFF4 in Tat-transactivation has been traced to a single amino acid variation between the two proteins, which causes them to enhance the affinity of Tat for P-TEFb, a key SEC component, with different efficiency. Finally, genome-wide analysis confirms that the genes regulated by AFF1-SEC and AFF4-SEC are largely non-overlapping and perform distinct functions. Thus, the SEC represents a family of related complexes that exist to increase the regulatory diversity and gene control options during transactivation of diverse cellular and viral genes.

AFF1 is a ubiquitous P-TEFb partner to enable Tat extraction of P-TEFb from 7SK snRNP and formation of SECs for HIV transactivation.

The positive transcription elongation factor b (P-TEFb) stimulates RNA polymerase elongation by inducing the transition of promoter proximally paused polymerase II into a productively elongating state. P-TEFb itself is regulated by reversible association with various transcription factors/cofactors to form several multisubunit complexes [e.g., the 7SK small nuclear ribonucleoprotein particle (7SK snRNP), the super elongation complexes (SECs), and the bromodomain protein 4 (Brd4)-P-TEFb complex] that constitute a P-TEFb network controlling cellular and HIV transcription. These complexes have been thought to share no components other than the core P-TEFb subunits cyclin-dependent kinase 9 (CDK9) and cyclin T (CycT, T1, T2a, and T2b). Here we show that the AF4/FMR2 family member 1 (AFF1) is bound to CDK9-CycT and is present in all major P-TEFb complexes and that the tripartite CDK9-CycT-AFF1 complex is transferred as a single unit within the P-TEFb network. By increasing the affinity of the HIV-encoded transactivating (Tat) protein for CycT1, AFF1 facilitates Tat's extraction of P-TEFb from 7SK snRNP and the formation of Tat-SECs for HIV transcription. Our data identify AFF1 as a ubiquitous P-TEFb partner and demonstrate that full Tat transactivation requires the complete SEC.

The BET bromodomain inhibitor JQ1 activates HIV latency through antagonizing Brd4 inhibition of Tat-transactivation.

Latent HIV reservoirs are the primary hurdle to eradication of infection. Identification of agents, pathways and molecular mechanisms that activate latent provirus may, in the presence of highly active antiretroviral therapy, permit clearance of infected cells by the immune system. Promoter-proximal pausing of RNA polymerase (Pol) II is a major rate-limiting step in HIV gene expression. The viral Tat protein recruits human Super Elongation Complex (SEC) to paused Pol II to overcome this limitation. Here, we identify the bromodomain protein Brd4 and its inhibition of Tat-transactivation as a major impediment to latency reactivation. Brd4 competitively blocks the Tat-SEC interaction on HIV promoter. The BET bromodomain inhibitor JQ1 dissociates Brd4 from the HIV promoter to allow Tat recruitment of SEC to stimulate HIV elongation. JQ1 synergizes with another latency activator prostratin, which promotes Pol II loading onto the viral promoter. Because JQ1 activates viral latency without inducing global T cell activation, this and other closely related compounds and their antagonization of Brd4 to promote Tat-SEC interaction merit further investigations as effective agents/strategies for eliminating latent HIV.

The ubiquitin ligase Siah1 controls ELL2 stability and formation of super elongation complexes to modulate gene transcription.

Super elongation complexes (SECs) contain two different transcription elongation factors, P-TEFb and ELL1/2, linked by the scaffolding protein AFF4 or AFF1. They stimulate the expression of both normal and disease-related genes, especially those of HIV or those involved in leukemogenesis. Among all SEC subunits, ELL2 is stoichiometrically limiting and uniquely regulated at the level of protein stability. Here we identify the RING domain protein Siah1, but not the homologous Siah2, as the E3 ubiquitin ligase for ELL2 polyubiquitination and proteasomal degradation. Siah1 cannot access and ubiquitinate ELL2 bound to AFF4, although, at high concentrations, it also degrades AFF4/1 to destroy SECs. Prostratin and HMBA, two well-studied activators of HIV transcription and latency, enhance ELL2 accumulation and SECs formation largely through decreasing Siah1 expression and ELL2 polyubiquitination. Given its importance in formation of SECs, the Siah1 ubiquitination pathway provides a fresh avenue for developing strategies to control disease-related transcription.

Analysis of white spot syndrome virus envelope protein complexome by two-dimensional blue native/SDS PAGE combined with mass spectrometry.

White spot syndrome virus (WSSV) is a large enveloped virus, but the organization of its envelope proteins remains largely unknown. In the present study, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and SDS-PAGE in combination with mass spectrometry to analyze the envelope protein complexome of WSSV. Our results show that the viral envelope consists of multi-protein complexes (MPCs). Within them, the envelope protein VP19 exists as a homotrimer, while another major envelope protein, VP28, mainly exists as a homotetramer. The most notable feature is that the majority of MPCs include VP26 and VP24, suggesting that these two proteins might serve as hub proteins to recruit low-abundance proteins to MPCs and play crucial roles in the process of protein complex formation. Furthermore, we found significant evidence for interactions between several low-abundance proteins, such as VP52B/VP38/VP33 and VP12/VP150. The result of this study may promote the further research on WSSV envelope assembly.