Tat controls transcriptional persistence of unintegrated HIV genome in primary human macrophages.

In HIV infected macrophages, a large population of viral genomes persists as the unintegrated form (uDNA) that is transcriptionally active. However, how this transcriptional activity is controlled remains unclear. In this report, we investigated whether Tat, the viral transactivator of transcription, is involved in uDNA transcription. We demonstrate that de novo Tat activity is generated from uDNA, and this uDNA-derived Tat (uTat) transactivates the uDNA LTR. In addition, uTat is required for the transcriptional persistence of uDNA that is assembled into repressive episomal minichromatin. In the absence of uTat, uDNA minichromatin is gradually silenced, but remains highly inducible by HDAC inhibitors (HDACi). Therefore, functionally, uTat antagonizes uDNA minichromatin repression to maintain persistent viral transcription in macrophages. uTat-mediated viral persistence may establish a viral reservoir in macrophages where uDNA were found to persist

PSGL-1 Inhibits the Incorporation of SARS-CoV and SARS-CoV-2 Spike Glycoproteins into Pseudovirions and Impairs Pseudovirus Attachment and Infectivity

P-selectin glycoprotein ligand-1 (PSGL-1) is a cell surface glycoprotein that binds to P-, E-, and L-selectins to mediate the tethering and rolling of immune cells on the surface of the endothelium for cell migration into inflamed tissues. PSGL-1 has been identified as an interferon-γ (INF-γ)-regulated factor that restricts HIV-1 infectivity, and has recently been found to possess broad-spectrum antiviral activities. Here we report that the expression of PSGL-1 in virus-producing cells impairs the incorporation of SARS-CoV and SARS-CoV-2 spike (S) glycoproteins into pseudovirions and blocks pseudovirus attachment and infection of target cells. These findings suggest that PSGL-1 may potentially inhibit coronavirus replication in PSGL-1+ cell

Correction: Phosphoproteomic analysis reveals Smad protein family activation following Rift Valley fever virus infection.

[This corrects the article DOI: 10.1371/journal.pone.0191983.]

Phosphoproteomic analysis reveals Smad protein family activation following Rift Valley fever virus infection

<div><p>Rift Valley fever virus (RVFV) infects both ruminants and humans leading to a wide variance of pathologies dependent on host background and age. Utilizing a targeted reverse phase protein array (RPPA) to define changes in signaling cascades after <i>in vitro</i> infection of human cells with virulent and attenuated RVFV strains, we observed high phosphorylation of Smad transcription factors. This evolutionarily conserved family is phosphorylated by and transduces the activation of TGF-β superfamily receptors. Moreover, we observed that phosphorylation of Smad proteins required active RVFV replication and loss of NSs impaired this activation, further corroborating the RPPA results. Gene promoter analysis of transcripts altered after RVFV infection identified 913 genes that contained a Smad-response element. Functional annotation of these potential Smad-regulated genes clustered in axonal guidance, hepatic fibrosis and cell signaling pathways involved in cellular adhesion/migration, calcium influx, and cytoskeletal reorganization. Furthermore, chromatin immunoprecipitation confirmed the presence of a Smad complex on the interleukin 1 receptor type 2 (IL1R2) promoter, which acts as a decoy receptor for IL-1 activation.</p></div

ChIP analysis of possible Smad-dependent transcripts.

<p>Lysates from mock, TGF-β activated, and MP12 infected were utilized for Smad4 (A) or methylated histone H3 lysine 4 (mH3K4; B) immunoprecipitations. Relative DNA abundance from immunoprecipitations was determined by qPCR. Signal over background normalization and fold enrichment was calculated for each condition. Bars represent means and standard deviations of four replicates. C) Levels of IL1R2, IL1RL1, and VAV3 RNA are analyzed by qRT-PCR. RNA lysates from HSAECs infected with MP12 (blue) or ZH548 (red) RVFV at an MOI 5 were utilized. Bars represent means and standard deviations of four replicates.</p

Potential Smad-regulated genes selected for ChIP analysis.

<p>Potential Smad-regulated genes selected for ChIP analysis.</p

Viral kinetics of virulent and attenuated RVFV viruses.

<p>A) Schematic depicting infection and harvest conditions for comparative analysis. HSAECs were infected with attenuated (MP12) or virulent (ZH548) RVFV at an MOI 5 using conditioned media. RVFV RNA replication (B), percentage of RVFV NP positivity (C), and infectious viral titers (D) were determined at the indicated time points (hours post infection (hpi)). The data are plotted as means with standard deviations: for panel (B) n = 3, (C) n = 6 and (D) n = 9. ** = P≤0.01 and **** = P≤0.0001.</p

Promoter analysis of RVFV RNASeq yields potential Smad-dependent promoters.

<p>A) Schematic depicting data-mining of RVFV RNASeq dataset for Smad-dependent promoters. B) Top ten significantly altered canonical pathways from IPA are shown for the virulent RVFV strain at 18hpi. The top axis indicates the statistical significance calculated using the right-tailed Fisher exact test. Threshold line represents significance cut-off at <i>p = 0</i>.<i>05</i>. Bars represent the level of significance, with orange and blue bars indicating whether the pathway is predicted to be activated or inhibited, respectively (z-score). Pathways where no prediction can be made are colored gray. The ratio (bottom axis) represents the number of molecules identified in a given pathway divided by total number of molecules that constitute that pathway.</p

siRNA knockdown of Smad1, -2 and 4 does not impact RVFV replication.

<p>HSAECs transfected with media alone (Mock) or siRNAs (75nM) directed against a nontargeting control (NTC), Smad1, -2, or -4 were infected with MP12 virus (MOI 0.1). Both extracellular media supernatants (A) and protein lysates (B,C) were harvested at 9 and 24hpi. A) Infectious viral titers were determined by plaque assay. Data plotted as a bar graph of the mean with standard deviation of four replicates. Protein lysates from single (B) and double knockdowns (C) were analyzed by western blot for actin, total Smad1, -2, and -4 expression.</p

ChIP analysis of possible Smad-dependent transcripts.

<p>Lysates from mock, TGF-β activated, and MP12 infected were utilized for Smad4 (A) or methylated histone H3 lysine 4 (mH3K4; B) immunoprecipitations. Relative DNA abundance from immunoprecipitations was determined by qPCR. Signal over background normalization and fold enrichment was calculated for each condition. Bars represent means and standard deviations of four replicates. C) Levels of IL1R2, IL1RL1, and VAV3 RNA are analyzed by qRT-PCR. RNA lysates from HSAECs infected with MP12 (blue) or ZH548 (red) RVFV at an MOI 5 were utilized. Bars represent means and standard deviations of four replicates.</p