Nirojini Sivachandran

MicroRNA-146 represses endothelial activation by inhibiting pro-inflammatory pathways

Activation of inflammatory pathways in the endothelium contributes to vascular diseases, including sepsis and atherosclerosis. We demonstrate that miR‐146a and miR‐146b are induced in endothelial cells upon exposure to pro‐inflammatory cytokines. Despite the rapid transcriptional induction of the miR‐146a/b loci, which is in part mediated by EGR‐3, miR‐146a/b induction is delayed and sustained compared to the expression of leukocyte adhesion molecules, and in fact coincides with the down‐regulation of inflammatory gene expression. We demonstrate that miR‐146 negatively regulates inflammation. Over‐expression of miR‐146a blunts endothelial activation, while knock‐down of miR‐146a/b in vitro or deletion of miR‐146a in mice has the opposite effect. MiR‐146 represses the pro‐inflammatory NF‐κB pathway as well as the MAP kinase pathway and downstream EGR transcription factors. Finally, we demonstrate that HuR, an RNA binding protein that promotes endothelial activation by suppressing expression of endothelial nitric oxide synthase (eNOS), is a novel miR‐146 target. Thus, we uncover an important negative feedback regulatory loop that controls pro‐inflammatory signalling in endothelial cells that may impact vascular inflammatory diseases.

Epstein-Barr Nuclear Antigen 1 Contributes to Nasopharyngeal Carcinoma through Disruption of PML Nuclear Bodies

Latent Epstein-Barr virus (EBV) infection is strongly associated with several cancers, including nasopharyngeal carcinoma (NPC), a tumor that is endemic in several parts of the world. We have investigated the molecular basis for how EBV latent infection promotes the development of NPC. We show that the viral EBNA1 protein, previously known to be required to maintain the EBV episomes, also causes the disruption of the cellular PML (promyelocytic leukemia) nuclear bodies (or ND10s). This disruption occurs both in the context of a native latent infection and when exogenously expressed in EBV-negative NPC cells and involves loss of the PML proteins. We also show that EBNA1 is partially localized to PML nuclear bodies in NPC cells and interacts with a specific PML isoform. PML disruption by EBNA1 requires binding to the cellular ubiquitin specific protease, USP7 or HAUSP, but is independent of p53. We further observed that p53 activation, DNA repair and apoptosis, all of which depend on PML nuclear bodies, were impaired by EBNA1 expression and that cells expressing EBNA1 were more likely to survive after induction of DNA damage. The results point to an important role for EBNA1 in the development of NPC, in which EBNA1-mediated disruption of PML nuclear bodies promotes the survival of cells with DNA damage.

Epstein-Barr Virus Nuclear Antigen 1 Hijacks the Host Kinase CK2 To Disrupt PML Nuclear Bodies

ABSTRACT Latent Epstein-Barr virus (EBV) infection is an important causative factor in the development of several cancers, including nasopharyngeal carcinoma (NPC). The one EBV protein expressed in the nucleus of NPC cells, EBNA1, has been shown to disrupt promyelocitic leukemia (PML) nuclear bodies (NBs) by inducing the degradation of PML proteins, leading to impaired DNA repair and increased cell survival. Although EBNA1-mediated PML disruption is likely to be an important factor in the development of NPC, little is known about its mechanism. We now show that an interaction between EBNA1 and the host CK2 kinase is crucial for EBNA1 to disrupt PML bodies and degrade PML proteins. EBNA1 increases the association of CK2 with PML proteins, thereby increasing the phosphorylation of PML proteins by CK2, a modification that is known to trigger the polyubiquitylation and degradation of PML. The interaction between EBNA1 and CK2 is direct and occurs through the β regulatory subunit of CK2 and EBNA1 amino acids 387 to 394. The binding of EBNA1 to the host ubiquitin specific protease USP7 has also been shown to be important for EBNA1-mediated PML disruption. We show that EBNA1 also increases the occupancy of USP7 at PML NBs and that CK2 and USP7 bind independently and simultaneously to EBNA1 to form a ternary complex. The combined results indicate that EBNA1 usurps two independent cellular pathways to trigger the loss of PML NBs.

Functions of the Epstein-Barr Virus EBNA1 Protein in Viral Reactivation and Lytic Infection

ABSTRACT EBNA1 is the only nuclear Epstein-Barr virus (EBV) protein expressed in both latent and lytic modes of infection. While EBNA1 is known to play several important roles in latent infection, the reason for its continued expression in lytic infection is unknown. Here we identified two roles for EBNA1 in the reactivation of latent EBV to the lytic cycle in epithelial cells. First, EBNA1 depletion in latently infected cells was shown to positively contribute to spontaneous EBV reactivation, showing that EBNA1 has a role in suppressing reactivation. Second, when the lytic cycle was induced, EBNA1 depletion decreased lytic gene expression and DNA amplification, showing that it positively contributed to lytic infection. Since we have previously shown that EBNA1 disrupts promyelocytic leukemia (PML) nuclear bodies, we investigated whether this function could account for the effects of EBNA1 on lytic infection by repeating the experiments with cells lacking PML proteins. In the absence of PML, EBNA1 did not promote lytic infection, indicating that the EBNA1-mediated PML disruption is responsible for promoting lytic infection. In keeping with this conclusion, PML silencing was found to be sufficient to induce the EBV lytic cycle. Finally, by generating cells with single PML isoforms, we showed that individual PML isoforms were sufficient to suppress EBV lytic reactivation, although PML isoform IV (PML IV) was ineffective because it was most efficiently degraded by EBNA1. Our results provide the first function for EBNA1 in lytic infection and show that EBNA1 interactions with PML IV lead to a loss of PML nuclear bodies (NBs) that promotes lytic infection.

Regulation of the EBNA1 Epstein-Barr Virus Protein by Serine Phosphorylation and Arginine Methylation

ABSTRACT The Epstein-Barr virus (EBV) EBNA1 protein is important for the replication and mitotic segregation of EBV genomes in latently infected cells and also activates the transcription of some of the viral latency genes. A Gly-Arg-rich region between amino acids 325 and 376 is required for both the segregation and transcriptional activation functions of EBNA1. Here we show that this region is modified by both arginine methylation and serine phosphorylation. Mutagenesis of the four potentially phosphorylated serines in this region indicated that phosphorylation of multiple serines contributes to the efficient segregation of EBV-based plasmids by EBNA1, at least in part by increasing EBNA1 binding to hEBP2. EBNA1 was also found to bind the arginine methyltransferases PRMT1 and PRMT5. Multiple arginines in the 325-376 region were methylated in vitro by PRMT1 and PRMT5, as was an N-terminal Gly-Arg-rich region between amino acids 41 and 50. EBNA1 was also shown to be methylated in vivo, predominantly in the 325-376 region. Treatment of cells with a methylation inhibitor or down-regulation of PRMT1 altered EBNA1 localization, resulting in the formation of EBNA1 rings around the nucleoli. The results indicate that EBNA1 function is influenced by both serine phosphorylation and arginine methylation.

Contributions of the Epstein-Barr Virus EBNA1 Protein to Gastric Carcinoma

ABSTRACT Approximately 10% of gastric carcinomas (GC) are comprised of cells latently infected with Epstein-Barr virus (EBV); however, the mechanism by which EBV contributes to the development of this malignancy is unclear. We have investigated the cellular effects of the only EBV nuclear protein expressed in GC, EBNA1, focusing on promyelocytic leukemia (PML) nuclear bodies (NBs), which play important roles in apoptosis, p53 activation, and tumor suppression. AGS GC cells infected with EBV were found to contain fewer PML NBs and less PML protein than the parental EBV-negative AGS cells, and these levels were restored by silencing EBNA1. Conversely, EBNA1 expression was sufficient to induce the loss of PML NBs and proteins in AGS cells. Consistent with PML functions, EBNA1 expression decreased p53 activation and apoptosis in response to DNA damage and resulted in increased cell survival. In addition, EBNA1 mutants unable to bind CK2 kinase or ubiquitin-specific protease 7 had decreased ability to induce PML loss and to interfere with p53 activation. PML levels in EBV-positive and EBV-negative GC biopsy specimens were then compared by immunohistochemistry. Consistent with the results in the AGS cells, EBV-positive tumors had significantly lower PML levels than EBV-negative tumors. The results indicate that EBV infection of GC cells leads to loss of PML NBs through the action of EBNA1, resulting in impaired responses to DNA damage and promotion of cell survival. Therefore, PML disruption by EBNA1 is one mechanism by which EBV may contribute to the development of gastric cancer.

Epstein-Barr Virus Nuclear Antigen 1 Replication and Segregation Functions in Nasopharyngeal Carcinoma Cell Lines

ABSTRACT Nasopharyngeal carcinomas (NPC) are usually Epstein-Barr virus (EBV) positive, but, with the exception of C666-1 cells, these cells lose the EBV genomes when grown in culture. Maintenance of EBV requires the viral EBV nuclear antigen 1 (EBNA1) protein, which ensures the replication and mitotic segregation of the genomes through interactions with OriP. Here we compare the abilities of C666-1 and NPC cells that have lost EBV genomes to replicate and segregate OriP plasmids. We found that either cell line can replicate and maintain OriP plasmids for extended periods under conditions where low levels of EBNA1 are expressed but that high EBNA1 levels selectively interfered with mitotic segregation.

Sexually transmitted infections and viral hepatitides in patients presenting for non-occupational HIV post-exposure prophylaxis: results of a prospective cohort study

Data evaluating the screening practices for viral hepatitides and sexually transmitted infections (STIs) in patients presenting for non-occupational HIV post-exposure prophylaxis (nPEP) care are limited. Screening practices and prevalences of viral hepatitides and STIs were evaluated in 126 patients presenting to a dedicated HIV prevention clinic for HIV nPEP. Three patients (2.4%) were diagnosed with chronic hepatitis C infection, 28 (22.2%) did not have surface antibodies in sufficient quantity to confer immunity to hepatitis B, and six (4.8%) were diagnosed with an STI. A multivariate regression model did not predict any demographic or clinical features predictive of HBV non-immunity. Beyond screening for HIV infection, evaluation for viral hepatitides and STIs is an important feature in the care of patients presenting for HIV nPEP.

Feasibility of HIV Pre-Exposure Prophylaxis as Part of Routine Care in Toronto, Canada.

INTRODUCTION The use of HIV Pre-Exposure Prophylaxis (PrEP) with oral tenofovir disproxil fumarate (TDF)/emtricitabine (FTC) (coformulated as Truvada) is effective in the prevention of HIV transmission through both sexual and injection drug exposures. The US Food and Drug Administration first approved TDF/FTC for PrEP in 2012, with comprehensive clinical practice guidelines released by the US Public Health Service in 2014. Currently, PrEP is not approved for this purpose by Health Canada and is prescribed off label. There are no published data about the use of PrEP in routine clinical practice in Canada, and little data of this sort have emerged from elsewhere. We aim to describe our experience with the use of PrEP in a cohort of Canadian patients receiving care in Toronto, Canada. We describe patient demographics, comorbidities, incidence of HIV, and other sexually transmitted infections (STIs), rates of follow-up, and reasons for discontinuation.

Barriers to the uptake of postexposure prophylaxis among Nairobi-based female sex workers.

Introduction:Female sex workers (FSWs) in sub-Saharan Africa are at a particularly high risk for HIV infection. Postexposure prophylaxis (PEP) is available as part of an HIV care and prevention program through dedicated FSW clinics in Nairobi, Kenya, but is underutilized. We evaluated PEP knowledge, access, and adherence among clinic attendees. Methods:An anonymous questionnaire was administered to unselected HIV-uninfected FSWs. Participants were dichotomized into high and low HIV risk categories based on self-reported sexual practices. Prior PEP use, knowledge, and adherence were then evaluated. Results:One hundred and thirty-four HIV-uninfected FSWs participated, with 64 (48%) categorized as being at high risk for HIV acquisition. High-risk FSWs were less likely to have heard of or accessed PEP than lower risk FSWs (37.5 vs. 58.6%, P = 0.014; and 21.9 vs. 40.6%, P = 0.019, respectively). Among higher risk FSWs, those who had accessed PEP were more likely to report treatment for a genital infection (71.4 vs. 42.0%, P = 0.049) or sex with an HIV-infected man (62.5 vs. 37.5%, P = 0.042) during the last 6 months. However, only 35.7% of high-risk women accessing PEP completed a full course of treatment, and noncompleters were more likely to report prior unprotected sex with an HIV-infected man (P = 0.023). Conclusion:Despite freely available PEP for Nairobi-based FSWs, women at highest risk were less likely to have heard of PEP, access PEP, or complete the full course of therapy once initiated. Program delivery needs to be improved to ensure that FSW most at risk are able to benefit from this resource.