A single point mutation in cyclin T1 eliminates binding to Hexim1, Cdk9 and RNA but not to AFF4 and enforces repression of HIV transcription

BACKGROUND: Human immunodeficiency virus (HIV) gene expression is primarily regulated at the step of transcription elongation. The viral Tat protein recruits the Positive Transcription Elongation Factor b (P-TEFb) and the Super Elongation Complex (SEC) to the HIV promoter and enhances transcription by host RNA polymerase II. RESULTS: To map residues in the cyclin box of cyclin T1 that mediate the binding of P-TEFb to its interacting host partners and support HIV transcription, a pool of N-terminal cyclin T1 mutants was generated. Binding and functional assays in cells identified specific positions in cyclin T1 that are important for (i) association of P-TEFb with Hexim1, Cdk9 and SEC/AFF4 (ii) supporting Tat-transactivation in murine cells and (iii) inhibition of basal and Tat-dependent HIV transcription in human cells. Significantly, a unique cyclin T1 mutant where a Valine residue at position 107 was mutated to Glutamate (CycT1-V107E) was identified. CycT1-V107E did not bind to Hexim1 or Cdk9, and also could not assemble on HIV TAR or 7SK-snRNA. However, it bound strongly to AFF4 and its association with HIV Tat was slightly impaired. CycT1-V107E efficiently inhibited HIV replication in human T cell lines and in CD4(+) primary cells, and enforced HIV transcription repression in T cell lines that harbor a transcriptionally silenced integrated provirus. CONCLUSIONS: This study outlines the mechanism by which CycT1-V107E mutant inhibits HIV transcription and enforces viral latency. It defines the importance of N-terminal residues of cyclin T1 in mediating contacts of P-TEFb with its transcription partners, and signifies the requirement of a functional P-TEFb and SEC in mediating HIV transcription

Fused in sarcoma silences HIV gene transcription and maintains viral latency through suppressing AFF4 gene activation

Background: The human immunodeficiency virus (HIV) cell reservoir is currently a main obstacle towards complete eradication of the virus. This infected pool is refractory to anti-viral therapy and harbors integrated proviruses that are transcriptionally repressed but replication competent. As transcription silencing is key for establishing the HIV reservoir, significant efforts have been made to understand the mechanism that regulate HIV gene transcription, and the role of the elongation machinery in promoting this step. However, while the role of the super elongation complex (SEC) in enhancing transcription activation of HIV is well established, the function of SEC in modulating viral latency is less defined and its cell partners are yet to be identified. Results: In this study we identify fused in sarcoma (FUS) as a partner of AFF4 in cells. FUS inhibits the activation of HIV transcription by AFF4 and ELL2, and silences overall HIV gene transcription. Concordantly, depletion of FUS elevates the occupancy of AFF4 and Cdk9 on the viral promoter and activates HIV gene transcription. Live cell imaging demonstrates that FUS co-localizes with AFF4 within nuclear punctuated condensates, which are disrupted upon treating cells with aliphatic alcohol. In HIV infected cells, knockout of FUS delays the gradual entry of HIV into latency, and similarly promotes viral activation in a T cell latency model that is treated with JQ1. Finally, effects of FUS on HIV gene transcription are also exhibited genome wide, where FUS mainly occupies gene promoters at transcription starting sites, while its knockdown leads to an increase in AFF4 and Cdk9 occupancy on gene promoters of FUS affected genes. Conclusions: Towards eliminating the HIV infected reservoir, understanding the mechanisms by which the virus persists in the face of therapy is important. Our observations show that FUS regulates both HIV and global gene transcription and modulates viral latency, thus can potentially serve as a target for future therapy that sets to reactivate HIV from its latent state.Israel Science Foundation [755/17]; Ben-Gurion University of the Negev, Israel; National Institute of Health [NS082376]; Office of the Director of the NIH [S10OD013237]; NIH [R21AI127274]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Genome-wide CRISPR knockout screen identifies ZNF304 as a silencer of HIV transcription that promotes viral latency.

Despite the widespread use of anti-retroviral therapy, human immunodeficiency virus (HIV) still persists in an infected cell reservoir that harbors transcriptionally silent yet replication-competent proviruses. While significant progress has been made in understanding how the HIV reservoir is established, transcription repression mechanisms that are enforced on the integrated viral promoter have not been fully revealed. In this study, we performed a whole-genome CRISPR knockout screen in HIV infected T cells to identify host genes that potentially promote HIV latency. Of several top candidates, the KRAB-containing zinc finger protein, ZNF304, was identified as the top hit. ZNF304 silences HIV gene transcription through associating with TRIM28 and recruiting to the viral promoter heterochromatin-inducing methyltransferases, including the polycomb repression complex (PRC) and SETB1. Depletion of ZNF304 expression reduced levels of H3K9me3, H3K27me3 and H2AK119ub repressive histone marks on the HIV promoter as well as SETB1 and TRIM28, ultimately enhancing HIV gene transcription. Significantly, ZNF304 also promoted HIV latency, as its depletion delayed the entry of HIV infected cells into latency. In primary CD4+ cells, ectopic expression of ZNF304 silenced viral transcription. We conclude that by associating with TRIM28 and recruiting host transcriptional repressive complexes, SETB1 and PRC, to the HIV promoter, ZNF304 silences HIV gene transcription and promotes viral latency

Functional Analysis of Spike from SARS-CoV-2 Variants Reveals the Role of Distinct Mutations in Neutralization Potential and Viral Infectivity

Enhanced viral transmission and escape from vaccine–elicited neutralizing antibodies drive worldwide spread of SARS-CoV-2 variants and promote disease progression. However, the impact of specific spike mutations that are carried by different viral variants on viral infectivity and neutralization sensitivity has not been completely defined. Here, we use pseudoviruses to assess the contribution of spike mutations within the Receptor Binding Domain (RBD) and the Furin Cleavage Site (FCS), and appear in circulating viral variants, on viral infectivity and neutralization potential against sera that was drawn from fully vaccinated individuals. Our functional analysis demonstrates that single, P681H, P681R or A701V–FCS mutations do not play a role in viral infectivity and neutralization potential. However, when in conjunction with the RBD–N501Y mutation, viral infectivity is enhanced. Similarly, combining the E484K–RBD mutation to the spike that carries FCS mutations reduces neutralization sensitivity with no effects on viral infectivity. Employing a similar approach onto the spike from Delta or Lota SARS-CoV-2 variants further reveals that specific RBD mutations affect neutralization sensitivity or viral infectivity differently. Our results validate the efficacy of the Pfizer third dose vaccine against Delta and Lota SARS-CoV-2 variants, and outline the significance of distinct RBD mutations in promoting viral infectivity and neutralization sensitivity to post–vaccination sera

Functional Mimetics of the HIV-1 CCR5 Co-Receptor Displayed on the Surface of Magnetic Liposomes.

Chemokine G protein coupled receptors, principally CCR5 or CXCR4, function as co-receptors for HIV-1 entry into CD4+ T cells. Initial binding of the viral envelope glycoprotein (Env) gp120 subunit to the host CD4 receptor induces a cascade of structural conformational changes that lead to the formation of a high-affinity co-receptor-binding site on gp120. Interaction between gp120 and the co-receptor leads to the exposure of epitopes on the viral gp41 that mediates fusion between viral and cell membranes. Soluble CD4 (sCD4) mimetics can act as an activation-based inhibitor of HIV-1 entry in vitro, as it induces similar structural changes in gp120, leading to increased virus infectivity in the short term but to virus Env inactivation in the long term. Despite promising clinical implications, sCD4 displays low efficiency in vivo, and in multiple HIV strains, it does not inhibit viral infection. This has been attributed to the slow kinetics of the sCD4-induced HIV Env inactivation and to the failure to obtain sufficient sCD4 mimetic levels in the serum. Here we present uniquely structured CCR5 co-receptor mimetics. We hypothesized that such mimetics will enhance sCD4-induced HIV Env inactivation and inhibition of HIV entry. Co-receptor mimetics were derived from CCR5 gp120-binding epitopes and functionalized with a palmitoyl group, which mediated their display on the surface of lipid-coated magnetic beads. CCR5-peptidoliposome mimetics bound to soluble gp120 and inhibited HIV-1 infectivity in a sCD4-dependent manner. We concluded that CCR5-peptidoliposomes increase the efficiency of sCD4 to inhibit HIV infection by acting as bait for sCD4-primed virus, catalyzing the premature discharge of its fusion potential

CCR5-peptidoliposomes enhance the ability of soluble CD4 mimetic to inhibit infection of R5-tropic HIV-1.

<p>CCR5-peptidoliposomes were co-incubated for 2 h with R5-tropic JRFL-pseudotyped HIV-1 (A); R5-tropic ADA-pseudotyped HIV-1 (B); or X4-tropic HXB2-pseudotyped HIV-1 (C), in the presence or absence of different concentrations of sCD4M48. Peptide-free magnetic liposomes (in the absence of sCD4M48) were used as control (set to 100% infectivity). The virus was separated from the beads by a magnetic field and TZM-HeLa-β-gal target cells were infected for 4 h. β-gal expression was carried out 48 h post infection. An unpaired <i>t</i>-test (two-tailed) was used to assess the significance of the difference in the means observed between the two groups indicated, <i>p</i> < 0.05. The data are mean ± S.E.M. calculated from three independent experiments each performed in duplicate.</p

CCR5-peptidoliposomes bind soluble recombinant gp120.

<p>Peptidoliposomes were incubated with 4 μM soluble recombinant His-tagged gp120 for 1 h at 37°C and the binding was assessed as described in the Materials and Methods. Mimetics used: mCD4 –CD4M48-PAL (1%); mCCR5-Nt – NT-2Y-CCR5-PAL (1%); mCCR5-ECL2 –ECL2-CCR5-2PAL (1%); mCCR5-Nt-3FSN–NT-3FSN-CCR5-PAL (1%); sCD4M48 –soluble M48 peptide (10 μM) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144043#pone.0144043.ref015" target="_blank">15</a>]. Control – peptide-free magnetic liposomes. <i>p</i> < 0.05. The data are mean ± S.E.M. calculated from three independent experiments each performed in triplicate.</p

Palmitoylated CCR5-peptidomimetics can be displayed on liposome surface.

<p>Peptidoliposomes were generated in the presence of increasing concentrations (molar %) of NT-2Y-CCR5-PAL (A) or ECL2-CCR5-2PAL (B) in the lipid mixture. (C) Peptidoliposomes were formed in the presence of 1% ECL2-CCR5-2PAL and the indicated concentrations of NT-2Y-CCR5-PAL in the lipid mixture. Peptidoliposomes were reacted with anti-CCR5 N-terminus polyclonal antibody (ab 7346, Abcam) (A) or with anti-CCR5 ECL2 (raised against a synthetic peptide 2D7-2SK [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144043#pone.0144043.ref044" target="_blank">44</a>]) polyclonal antibody (ab 36818, Abcam) (B and C), followed by a secondary HRP-conjugated antibody (ab 7090, Abcam) and analyzed as described in the Materials and Methods. Control – peptide-free magnetic liposomes. The data are mean ± S.E.M. calculated from three independent experiments each performed in triplicate.</p

HIV-1 incubation with CCR5-peptidoliposomes does not deplete viral loads.

<p>JRFL-pseudotyped HIV-1 was incubated for 2 h with peptidoliposomes in the presence or absence of M48 peptide–sCD4M48 (10 μM). CCR5-Beads - peptidoliposomes containing 5% each of NT-3FSN-CCR5-PAL and ECL2-CCR5-2PAL; (CD4+CCR5)-Beads - peptidoliposomes containing 5% each of CD4M48-PAL, ECL2-CCR5-2PAL and NT-3FSN-CCR5-PAL. At the end of the incubation, the supernatant and liposome fractions were analyzed by ELISA for the presence of HIV-p24 antigen as described in the Materials and Methods. Peptide-free magnetic liposomes were used as the control (p24 count in the supernatant set to 100%). The data are mean ± S.E.M. calculated from three independent experiments each performed in duplicate.</p

Magnetic liposome population is homogeneous.

<p>The magnetic liposomes were prepared using a lipid mixture POPC:POPE:DMPA (molar ratio 6:3:1) containing 1% Biotinyl-DOPE in the presence (black peak) or absence (white peak) of 1% Rhodamine–DOPE, and analyzed by FACS as described in the Materials and Methods. FL1-H designates the height of the photon peak obtained by using a 525/50 band pass filter (FL1). The figure shows the data for an experiment representative of two similar experiments.</p