HIV latency and integration site placement in five cell-based models

BACKGROUND: HIV infection can be treated effectively with antiretroviral agents, but the persistence of a latent reservoir of integrated proviruses prevents eradication of HIV from infected individuals. The chromosomal environment of integrated proviruses has been proposed to influence HIV latency, but the determinants of transcriptional repression have not been fully clarified, and it is unclear whether the same molecular mechanisms drive latency in different cell culture models. RESULTS: Here we compare data from five different in vitro models of latency based on primary human T cells or a T cell line. Cells were infected in vitro and separated into fractions containing proviruses that were either expressed or silent/inducible, and integration site populations sequenced from each. We compared the locations of 6,252 expressed proviruses to those of 6,184 silent/inducible proviruses with respect to 140 forms of genomic annotation, many analyzed over chromosomal intervals of multiple lengths. A regularized logistic regression model linking proviral expression status to genomic features revealed no predictors of latency that performed better than chance, though several genomic features were significantly associated with proviral expression in individual models. Proviruses in the same chromosomal region did tend to share the same expressed or silent/inducible status if they were from the same cell culture model, but not if they were from different models. CONCLUSIONS: The silent/inducible phenotype appears to be associated with chromosomal position, but the molecular basis is not fully clarified and may differ among in vitro models of latency

Histone H3.3 beyond cancer: Germline mutations in Histone 3 Family 3A and 3B cause a previously unidentified neurodegenerative disorder in 46 patients

Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation

Spliceosome malfunction causes neurodevelopmental disorders with overlapping features

Pre-mRNA splicing is a highly coordinated process. While its dysregulation has been linked to neurological deficits, our understanding of the underlying molecular and cellular mechanisms remains limited. We implicated pathogenic variants in U2AF2 and PRPF19, encoding spliceosome subunits in neurodevelopmental disorders (NDDs), by identifying 46 unrelated individuals with 23 de novo U2AF2 missense variants (including 7 recurrent variants in 30 individuals) and 6 individuals with de novo PRPF19 variants. Eight U2AF2 variants dysregulated splicing of a model substrate. Neuritogenesis was reduced in human neurons differentiated from human pluripotent stem cells carrying two U2AF2 hyper-recurrent variants. Neural loss of function (LoF) of the Drosophila orthologs U2af50 and Prp19 led to lethality, abnormal mushroom body (MB) patterning, and social deficits, which were differentially rescued by wild-type and mutant U2AF2 or PRPF19. Transcriptome profiling revealed splicing substrates or effectors (including Rbfox1, a third splicing factor), which rescued MB defects in U2af50deficient flies. Upon reanalysis of negative clinical exomes followed by data sharing, we further identified 6 patients with NDD who carried RBFOX1 missense variants which, by in vitro testing, showed LoF. Our study implicates 3 splicing factors as NDD-causative genes and establishes a genetic network with hierarchy underlying human brain development and function

Cell-to-Cell Transmission Can Overcome Multiple Donor and Target Cell Barriers Imposed on Cell-Free HIV

<div><p>Virus transmission can occur either by a cell-free mode through the extracellular space or by cell-to-cell transmission involving direct cell-to-cell contact. The factors that determine whether a virus spreads by either pathway are poorly understood. Here, we assessed the relative contribution of cell-free and cell-to-cell transmission to the spreading of the human immunodeficiency virus (HIV). We demonstrate that HIV can spread by a cell-free pathway if all the steps of the viral replication cycle are efficiently supported in highly permissive cells. However, when the cell-free path was systematically hindered at various steps, HIV transmission became contact-dependent. Cell-to-cell transmission overcame barriers introduced in the donor cell at the level of gene expression and surface retention by the restriction factor tetherin. Moreover, neutralizing antibodies that efficiently inhibit cell-free HIV were less effective against cell-to-cell transmitted virus. HIV cell-to-cell transmission also efficiently infected target T cells that were relatively poorly susceptible to cell-free HIV. Importantly, we demonstrate that the donor and target cell types influence critically the extent by which cell-to-cell transmission can overcome each barrier. Mechanistically, cell-to-cell transmission promoted HIV spread to more cells and infected target cells with a higher proviral content than observed for cell-free virus. Our data demonstrate that the frequently observed contact-dependent spread of HIV is the result of specific features in donor and target cell types, thus offering an explanation for conflicting reports on the extent of cell-to-cell transmission of HIV.</p> </div

Directly Infected Resting CD4+T Cells Can Produce HIV Gag without Spreading Infection in a Model of HIV Latency

<div><p>Despite the effectiveness of highly active antiretroviral therapy (HAART) in treating individuals infected with HIV, HAART is not a cure. A latent reservoir, composed mainly of resting CD4+T cells, drives viral rebound once therapy is stopped. Understanding the formation and maintenance of latently infected cells could provide clues to eradicating this reservoir. However, there have been discrepancies regarding the susceptibility of resting cells to HIV infection <em>in vitro</em> and <em>in vivo</em>. As we have previously shown that resting CD4+T cells are susceptible to HIV integration, we asked whether these cells were capable of producing viral proteins and if so, why resting cells were incapable of supporting productive infection. To answer this question, we spinoculated resting CD4+T cells with or without prior stimulation, and measured integration, transcription, and translation of viral proteins. We found that resting cells were capable of producing HIV Gag without supporting spreading infection. This block corresponded with low HIV envelope levels both at the level of protein and RNA and was not an artifact of spinoculation. The defect was reversed upon stimulation with IL-7 or CD3/28 beads. Thus, a population of latent cells can produce viral proteins without resulting in spreading infection. These results have implications for therapies targeting the latent reservoir and suggest that some latent cells could be cleared by a robust immune response.</p> </div

The actin cytoskeleton of Jurkat cells presents a barrier to cell-free HIV infection.

<p>(<b>A, B</b>) MT4, Jurkat and primary CD4 T cells were inoculated with concentrated cell-free HIV_NL4-3-GLuc by spinoculation and incubated at 37°C in the presence or absence of increasing concentrations of latrunculin-A (Lat-A) or japlakinolide (Jas)(µM). Luciferase activity was measured 36 h post-inoculation. Data were normalized to DMSO control. Error bars represent the standard error of the mean from 2 experiments. (<b>C</b>) Phalloidin staining of untreated, Lat-A-, and Jas-treated cells. Cells were exposed to Lat-A (1 µM for MT4 and 0.5 µM for Jurkat) or Jas (0.5 µM for MT-4 and 0.0625 µM for Jurkat) for 1 h at 37°C. Note that phalloidin competes with Jas for binding to polymerized actin and further dilution of drug was required to observe actin staining in Jurkat cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053138#pone.0053138-Bubb1" target="_blank">[76]</a>. Size bars correspond to 10<b> </b>µm. (<b>D</b>) Viral binding was measured by α-p24-ELISA after spinoculating cells in the presence or absence of 1 µM Lat-A or Jas. Error bars represent the standard deviation from 3 measurements. (<b>E</b>) Late reverse transcription (RT) was measured by Q-PCR from cells treated with 1 µM of Lat-A or Jas. Error bars represent the standard deviation of 3 late RT measurements. (<b>F, G</b>) A co-culture experiment as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053138#pone-0053138-g005" target="_blank">Figure 5B</a> was performed in the presence of increasing concentrations of Lat-A or Jas (µM). Error bars represent the standard error of the mean from 2 experiments.</p

The relative contribution of cell-free to co-culture mediated transmission is affected by the donor cell type.

<p>Different donor cells (HEK293, HeLa, Jurkat) were co-cultured with HeLa cells expressing CD4/CXCR4, MT4 cells and Jurkat cells. The efficiency of virus transmission in the cell-free mode and in the co-culture-dependent mode was compared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053138#pone-0053138-g001" target="_blank">Figure 1A</a>. Data for HEK293 donor cells are as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053138#pone-0053138-g005" target="_blank">Figure 5B</a>. Error bars represent the standard error of the mean from 2 experiments.</p

Infected resting CD4+T cells can produce HIV Gag.

<p>Bulk unstimulated CD4+T cells were spinoculated and cultured in the presence of the protease inhibitor, saquinavir. Gag protein was measured in the endogenously activated (HLA-DR+, CD25+, or CD69+) and resting (HLA-DR−,CD25−,and CD69−) cells based on activation marker expression at 0, 24, 48, 72, and 96 hours post infection. Control cells were treated with the reverse transcriptase (RT) inhibitor, efavirenz, to establish background protein levels. An average of 3 experiments in 3 different donors is shown (A). In B, purified resting or CCL19 treated cells were infected with HIV with or without spinoculation at 1200×g. Controls and gates were made as in A. Cells were gated on the activation marker negative (HLA-DR−,CD25−,CD69−) population. Approximately 10,000 events were collected for spinoculated samples while approximately 100,000 events were collected for cells that were not spinoculated. Data is a representative of 2 experiments in 2 different donors.</p

Gag expressing resting CD4+T cells remain in a latent state.

<p>In A, purified resting CD4+T cells were spinoculated with NL4-3 (MOI of 3) and cultured in the presence or absence of saquinavir for 4 days. Intracellular Gag was measured 96 hours post infection. Control cells were treated as in A and gates were set using an efavirenz treated control. Then resting cells cultured without saquinavir were stimulated with PHA+100 U/mL IL-2 for 48 hours in the presence or absence of saquinavir. Intracellular Gag was again measured. In B, cells were purified and treated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002818#ppat-1002818-g001" target="_blank">Figure 1</a> but infected at a MOI of 0.2. After infection, half the cells were treated with saquinavir. Cells were collected at 72 hours for CD3/28 activated cells and at 7 days post infection for all other cells. Total DNA was measured in both fractions of cells. The average of 3 experiments with 3 different donors is shown. In C, resting and CD3/28 activated cells were spinoculated with HIV (MOI of 3). Supernatant was collected 96 hours post infection. CEMss-GFP cells were spinoculated with the collected supernatant from resting and activated cells. An efavirenz control was used to determine background GFP levels. A representative experiment is shown in C. An average of 2 experiments in 2 different donors is shown in D. Error bars represent the standard error of the measurements. *Statistically significant at p<0.05 level. ND = not detectable.</p

Size of barriers experimentally introduced into the cell-free path of HIV.

<p>By interfering with the cell-free path of HIV, these barriers tilt virus transmission towards contact-dependent modes. See text for details.</p