ResearchPad - viruses-and-cancer https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Human papillomavirus E7 binds Oct4 and regulates its activity in HPV-associated cervical cancers]]> https://www.researchpad.co/article/elastic_article_14755 The transcription factor Oct4 with well-known roles in embryogenesis, pluripotency and cellular reprogramming has recently been found to be expressed in several types of somatic tumors. Even though its role in cancer remains controversial, we provide evidence that Oct4 is expressed in cervical cancer tissues and cancer cell lines. The viral oncogenes of the Human Papillomavirus significantly elevate Oct4 expression both in normal and cancer cells, likely through transcriptional upregulation. While the expression levels of Oct4 in cancer are low compared to those seen in stem cells, our results suggest that they are still consequential to cell proliferation, self-renewal, and migration. We demonstrate a physical interaction of the E7 oncoprotein with Oct4, mapping to the CR3 region of E7, which correlates to a distinct Oct4 transcriptional output. Introduction of E7 into HPV(-) cells and immortalised human keratinocytes led to transcriptional and phenotypic changes, which mimicked results in HPV(+) cells. These insights provide a plausible mechanism and consequences for a long-suspected interaction.

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<![CDATA[Convergent perturbation of the human domain-resolved interactome by viruses and mutations inducing similar disease phenotypes]]> https://www.researchpad.co/article/5c6dc9afd5eed0c484529ff0

An important goal of systems medicine is to study disease in the context of genetic and environmental perturbations to the human interactome network. For diseases with both genetic and infectious contributors, a key postulate is that similar perturbations of the human interactome by either disease mutations or pathogens can have similar disease consequences. This postulate has so far only been tested for a few viral species at the level of whole proteins. Here, we expand the scope of viral species examined, and test this postulate more rigorously at the higher resolution of protein domains. Focusing on diseases with both genetic and viral contributors, we found significant convergent perturbation of the human domain-resolved interactome by endogenous genetic mutations and exogenous viral proteins inducing similar disease phenotypes. Pan-cancer, pan-oncovirus analysis further revealed that domains of human oncoproteins either physically targeted or structurally mimicked by oncoviruses are enriched for cancer driver rather than passenger mutations, suggesting convergent targeting of cancer driver pathways by diverse oncoviruses. Our study provides a framework for high-resolution, network-based comparison of various disease factors, both genetic and environmental, in terms of their impacts on the human interactome.

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<![CDATA[Reining in the CD8+ T cell: Respiratory virus infection and PD-1-mediated T-cell impairment]]> https://www.researchpad.co/article/5c37b7bad5eed0c484490aa4 ]]> <![CDATA[Influence of ND10 Components on Epigenetic Determinants of Early KSHV Latency Establishment]]> https://www.researchpad.co/article/5989db09ab0ee8fa60bc9576

We have previously demonstrated that acquisition of intricate patterns of activating (H3K4me3, H3K9/K14ac) and repressive (H3K27me3) histone modifications is a hallmark of KSHV latency establishment. The precise molecular mechanisms that shape the latent histone modification landscape, however, remain unknown. Promyelocytic leukemia nuclear bodies (PML-NB), also called nuclear domain 10 (ND10), have emerged as mediators of innate immune responses that can limit viral gene expression via chromatin based mechanisms. Consequently, although ND10 functions thus far have been almost exclusively investigated in models of productive herpesvirus infection, it has been proposed that they also may contribute to the establishment of viral latency. Here, we report the first systematic study of the role of ND10 during KSHV latency establishment, and link alterations in the subcellular distribution of ND10 components to a temporal analysis of histone modification acquisition and host cell gene expression during the early infection phase. Our study demonstrates that KSHV infection results in a transient interferon response that leads to induction of the ND10 components PML and Sp100, but that repression by ND10 bodies is unlikely to contribute to KSHV latency establishment. Instead, we uncover an unexpected role for soluble Sp100 protein, which is efficiently and permanently relocalized from nucleoplasmic and chromatin-associated fractions into the insoluble matrix. We show that LANA expression is sufficient to induce Sp100 relocalization, likely via mediating SUMOylation of Sp100. Furthermore, we demonstrate that depletion of soluble Sp100 occurs precisely when repressive H3K27me3 marks first accumulate on viral genomes, and that knock-down of Sp100 (but not PML or Daxx) facilitates H3K27me3 acquisition. Collectively, our data support a model in which non-ND10 resident Sp100 acts as a negative regulator of polycomb repressive complex-2 (PRC2) recruitment, and suggest that KSHV may actively escape ND10 silencing mechanisms to promote establishment of latent chromatin.

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<![CDATA[LANA Binds to Multiple Active Viral and Cellular Promoters and Associates with the H3K4Methyltransferase hSET1 Complex]]> https://www.researchpad.co/article/5989dae3ab0ee8fa60bbc56a

Kaposi's sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus associated with KS and two lymphoproliferative diseases. Recent studies characterized epigenetic modification of KSHV episomes during latency and determined that latency-associated genes are associated with H3K4me3 while most lytic genes are associated with the silencing mark H3K27me3. Since the latency-associated nuclear antigen (LANA) (i) is expressed very early after de novo infection, (ii) interacts with transcriptional regulators and chromatin remodelers, and (iii) regulates the LANA and RTA promoters, we hypothesized that LANA may contribute to the establishment of latency through epigenetic control. We performed a detailed ChIP-seq analysis in cells of lymphoid and endothelial origin and compared H3K4me3, H3K27me3, polII, and LANA occupancy. On viral episomes LANA binding was detected at numerous lytic and latent promoters, which were transactivated by LANA using reporter assays. LANA binding was highly enriched at H3K4me3 peaks and this co-occupancy was also detected on many host gene promoters. Bioinformatic analysis of enriched LANA binding sites in combination with biochemical binding studies revealed three distinct binding patterns. A small subset of LANA binding sites showed sequence homology to the characterized LBS1/2 sequence in the viral terminal repeat. A large number of sites contained a novel LANA binding motif (TCCAT)3 which was confirmed by gel shift analysis. Third, some viral and cellular promoters did not contain LANA binding sites and are likely enriched through protein/protein interaction. LANA was associated with H3K4me3 marks and in PEL cells 86% of all LANA bound promoters were transcriptionally active, leading to the hypothesis that LANA interacts with the machinery that methylates H3K4. Co-immunoprecipitation demonstrated LANA association with endogenous hSET1 complexes in both lymphoid and endothelial cells suggesting that LANA may contribute to the epigenetic profile of KSHV episomes.

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<![CDATA[Larger Mammalian Body Size Leads to Lower Retroviral Activity]]> https://www.researchpad.co/article/5989d9feab0ee8fa60b72f94

Retroviruses have been infecting mammals for at least 100 million years, leaving descendants in host genomes known as endogenous retroviruses (ERVs). The abundance of ERVs is partly determined by their mode of replication, but it has also been suggested that host life history traits could enhance or suppress their activity. We show that larger bodied species have lower levels of ERV activity by reconstructing the rate of ERV integration across 38 mammalian species. Body size explains 37% of the variance in ERV integration rate over the last 10 million years, controlling for the effect of confounding due to other life history traits. Furthermore, 68% of the variance in the mean age of ERVs per genome can also be explained by body size. These results indicate that body size limits the number of recently replicating ERVs due to their detrimental effects on their host. To comprehend the possible mechanistic links between body size and ERV integration we built a mathematical model, which shows that ERV abundance is favored by lower body size and higher horizontal transmission rates. We argue that because retroviral integration is tumorigenic, the negative correlation between body size and ERV numbers results from the necessity to reduce the risk of cancer, under the assumption that this risk scales positively with body size. Our model also fits the empirical observation that the lifetime risk of cancer is relatively invariant among mammals regardless of their body size, known as Peto's paradox, and indicates that larger bodied mammals may have evolved mechanisms to limit ERV activity.

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<![CDATA[Kaposi's Sarcoma-Associated Herpesvirus-Encoded LANA Down-Regulates IL-22R1 Expression through a Cis-Acting Element within the Promoter Region]]> https://www.researchpad.co/article/5989da84ab0ee8fa60b9ba15

Kaposi's sarcoma-associated herpesvirus (KSHV) is considered to be a necessary, but not sufficient, causal agent of Kaposi's sarcoma (KS). All forms of KS are characterized by the proliferation of spindle-shaped cells, and most (>90%) spindle cells from KS lesions are latently infected with KSHV. During KSHV latency, only a few viral genes are expressed. Among those latent genes, the ORF 73 gene encodes the latency-associated nuclear antigen (LANA), which is critical for the establishment and maintenance of the latent KSHV infection. Much evidence suggests that many cytokines can increase the frequency and aggressiveness of KS. In this study, a microarray analysis of KS and normal tissues revealed that multiple cytokines and cytokine receptors are regulated by KSHV latent infection. Of special interest, IL-22R1 transcript level was found to be down-regulated in the KS tissue. To study the possible regulation of IL-22R1 by LANA, the IL-22R1 promoter was constructed and found to contain a LANA-binding site (LBS). LANA was demonstrated to down-regulate IL-22R1 expression via direct binding to the LBS located within the IL-22R1 promoter region. Furthermore, KSHV latently infected cells showed an impaired response to IL-22 stimulation. These results suggest that LANA can regulate host factor expression by directly binding to a cis-acting element within the factor's promoter to benefit latent viral infection and suppression of the antiviral immune response.

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<![CDATA[Gender Differences in Clinical Presentation and Outcomes of Epidemic Kaposi Sarcoma in Uganda]]> https://www.researchpad.co/article/5989da35ab0ee8fa60b8604d

Introduction

The incidence of Kaposi sarcoma (KS) has increased dramatically among women in sub-Saharan Africa since the onset of the HIV pandemic, but data on KS disease in women are limited. To identify gender-related differences in KS presentation and outcomes, we evaluated the clinical manifestations and response in men and women with AIDS-associated KS in Uganda.

Methods and Findings

HIV-infected adults with KS attending the Infectious Diseases Institute (IDI) and Uganda Cancer Institute (UCI) in Kampala, Uganda between 2004 and 2006 were included in a retrospective cohort. Evaluation of KS presentation was based on the clinical features described at the initial KS visit. Response was evaluated as the time to “improvement”, as defined by any decrease in lesion size, lesion number, or edema. The cohort consisted of 197 adults with HIV and KS: 55% (108/197) were women. At presentation, the median CD4 T-cell count was significantly lower in women (58 cells/mm3; IQR 11–156 cells/mm3) than men (124 cells/mm3; IQR 22–254 cells/mm3) (p = 0.02). Women were more likely than men to present with lesions of the face (OR 2.8, 95% CI, 1.4, 5.7; p = 0.005) and hard palate (OR 2.0, 95% CI, 1.1, 3.7; p = 0.02), and were less likely than men to have lower extremity lesions (OR 0.54, 95% CI, 0.3, 0.99; p = 0.05). Women were less likely than men to demonstrate clinical improvement (HR = 0.52, CI 0.31, 0.88; p = 0.01) in multivariate analysis.

Conclusions

The clinical presentation and response of KS differs between men and women in Uganda. These data suggest that gender affects the pathophysiology of KS, which may have implications for the prevention, diagnosis, and treatment of KS in both men and women. Prospective studies are needed to identify predictors of response and evaluate efficacy of treatment in women with KS, particularly in Africa where the disease burden is greatest.

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<![CDATA[Common Commensal Cancer Viruses]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdcef2 ]]> <![CDATA[PLoS Biology Issue Image | Vol. 16(6) June 2018]]> https://www.researchpad.co/article/5b4a0356463d7e3e7a971179

Structure of the herpes simplex virus portal-vertex

The herpesvirus family includes many important human pathogens such as herpes simplex viruses that cause cold-sores and human cytomegalovirus, a major cause of congenital abnormalities; several herpes viruses are known to cause cancer. Herpes viruses assemble enveloped virus particles (virions) that incorporate a large DNA-containing icosahedral capsid. Virion assembly commences in the nucleus of an infected cell, where the viral genome is pumped into preassembled capsids by a portal motor that is located at a unique 5-fold symmetry axis—the portal-vertex. This study by McElwee et al. used cryogenic electron microscopy and 3D image reconstruction to solve the structure of the portal-vertex of herpes simplex virus type 1 at 8 Å resolution. Their structure reveals the presence of several previously unknown features including a novel pentameric assembly that exhibits a coiled-coil motif comprising two α-helices. The image shows a close-up view of the portal-vertex, including the portal (mauve), the pentameric portal-vertex protein (purple), and the periportal triplex-like density (magenta).

Image Credit: 10.1371/journal.pbio.2006191

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<![CDATA[Lessons from Reverse Translation]]> https://www.researchpad.co/article/5989da06ab0ee8fa60b75b7b ]]> <![CDATA[Impact of HIV Infection and Kaposi Sarcoma on Human Herpesvirus-8 Mucosal Replication and Dissemination in Uganda]]> https://www.researchpad.co/article/5989da35ab0ee8fa60b8603b

Introduction

Kaposi sarcoma (KS) is the leading cause of cancer in Uganda and occurs in people with and without HIV. Human herpesvirus-8 (HHV-8) replication is important both in transmission of HHV-8 and progression to KS. We characterized the sites and frequency of HHV-8 detection in Ugandans with and without HIV and KS.

Methods

Participants were enrolled into one of four groups on the basis of HIV and KS status (HIV negative/KS negative, HIV positive/KS negative, HIV negative/KS positive, and HIV positive/KS positive). Participants collected oral swabs daily and clinicians collected oral swabs, anogenital swabs, and plasma samples weekly over 4 weeks. HHV-8 DNA at each site was quantified by polymerase chain reaction (PCR).

Results

78 participants collected a total of 2063 orals swabs and 358 plasma samples. Of these, 428 (21%) oral swabs and 96 (27%) plasma samples had detectable HHV-8 DNA. HHV-8 was detected more frequently in both the oropharynx of persons with KS (24 (57%) of 42 persons with KS vs. 8 (22%) of 36 persons without, p = 0.002) and the peripheral blood (30 (71%) of 42 persons with KS vs. 8 (22%) of 36 persons without, p<0.001). In a multivariate model, HHV-8 viremia was more frequent among men (IRR = 3.3, 95% CI = 1.7–6.2, p<0.001), persons with KS (IRR = 3.9, 95% CI = 1.7–9.0, p = 0.001) and persons with HIV infection (IRR = 1.7, 95% CI = 1.0–2.7, p = 0.03). Importantly, oral HHV-8 detection predicted the subsequent HHV-8 viremia. HHV-8 viremia was significantly more common when HHV-8 DNA was detected from the oropharynx during the week prior than when oral HHV-8 was not detected (RR = 3.3, 95% CI = 1.8–5.9 p<0.001). Genital HHV-8 detection was rare (9 (3%) of 272 swabs).

Conclusions

HHV-8 detection is frequent in the oropharynx and peripheral blood of Ugandans with endemic and epidemic KS. Replication at these sites is highly correlated, and viremia is increased in men and those with HIV. The high incidence of HHV-8 replication at multiple anatomic sites may be an important factor leading to and sustaining the high prevalence of KS in Uganda.

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<![CDATA[Direct Repeat 6 from Human Herpesvirus-6B Encodes a Nuclear Protein that Forms a Complex with the Viral DNA Processivity Factor p41]]> https://www.researchpad.co/article/5989da0aab0ee8fa60b77301

The SalI-L fragment from human herpesvirus 6A (HHV-6A) encodes a protein DR7 that has been reported to produce fibrosarcomas when injected into nude mice, to transform NIH3T3 cells, and to interact with and inhibit the function of p53. The homologous gene in HHV-6B is dr6. Since p53 is deregulated in both HHV-6A and -6B, we characterized the expression of dr6 mRNA and the localization of the translated protein during HHV-6B infection of HCT116 cells. Expression of mRNA from dr6 was inhibited by cycloheximide and partly by phosphonoacetic acid, a known characteristic of herpesvirus early/late genes. DR6 could be detected as a nuclear protein at 24 hpi and accumulated to high levels at 48 and 72 hpi. DR6 located in dots resembling viral replication compartments. Furthermore, a novel interaction between DR6 and the viral DNA processivity factor, p41, could be detected by confocal microscopy and by co-immunoprecipitation analysis. In contrast, DR6 and p53 were found at distinct subcellular locations. Together, our data imply a novel function of DR6 during HHV-6B replication.

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<![CDATA[Copy Number Variation and Differential Expression of a Protective Endogenous Retrovirus in Sheep]]> https://www.researchpad.co/article/5989da39ab0ee8fa60b876e7

The Jaagsiekte sheep retrovirus exJSRV and its endogenous counterpart enJSRV co-exist in sheep. exJSRV, a betaretrovirus, is the etiological agent of ovine pulmonary adenocarcinoma, and it has been demonstrated in vitro that an enJSRV Gag variant bearing the R-to-W amino acid change at position 21 was able to block exJSRV budding from the cells, providing a potential protective role for the host. In this work, we developed a fast mutation detection assay based on the oligo ligation assay (OLA) that permits the quantification of the relative proportions of the R21 and W21 Gag variants present in individual genomes and in cDNA obtained from normal and exJSRV-induced lung tumors. We have shown that the W21/R21 ratio is variable within and between breeds. We also describe for the first time that putative protecting enJSRV variants were expressed in alveolar type II cells (AECII), the major target of exJSRV.

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<![CDATA[XMRV Induces Cell Migration, Cytokine Expression and Tumor Angiogenesis: Are 22Rv1 Cells a Suitable Prostate Cancer Model?]]> https://www.researchpad.co/article/5989da47ab0ee8fa60b8c2ad

22Rv1 is a common prostate cancer cell line used in xenograft mouse experiments as well as in vitro cell culture assays to study aspects of prostate cancer tumorigenesis. Recently, this cell line was shown to harbor multiple copies of a gammaretrovirus, called XMRV, integrated in its genome. While the original prostate cancer xenograft CWR22 is free of any retrovirus, subsequently generated cell lines 22Rv1 and CWR-R1, carry this virus and additionally shed infectious gammaretroviral particles in their supernatant. Although XMRV most likely was generated by recombination events in cell culture this virus has been demonstrated to infect human cells in vitro and 22Rv1 as well as CWR-R1 cells are now considered biosafety 2 reagents. Here, we demonstrate that 22Rv1 cells with reduced retroviral transcription show reduced tumor angiogenesis and increased necrosis of the primary tumor derived from xenografted cells in scid mice when compared to the parental cell line. The presence of XMRV transcripts significantly increases secretion of osteopontin (OPN), CXCL14, IL13 and TIMP2 in 22Rv1 cells. Furthermore, these data are supported by in vitro cell invasion and differentiation assays. Collectively, our data suggest that the presence of XMRV transcripts at least partially contributes to 22Rv1 characteristics observed in vitro and in vivo with regard to migration, invasion and tumor angiogenesis. We propose that data received with 22Rv1 cells or equivalent cells carrying xenotropic gammaretroviruses should be carefully controlled including other prostate cancer cell lines tested for viral sequences.

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<![CDATA[HTLV-1 Propels Thymic Human T Cell Development in “Human Immune System” Rag2-/- gamma c-/- Mice]]> https://www.researchpad.co/article/5989db0bab0ee8fa60bca1f7

Alteration of early haematopoietic development is thought to be responsible for the onset of immature leukemias and lymphomas. We have previously demonstrated that TaxHTLV-1 interferes with ß-selection, an important checkpoint of early thymopoiesis, indicating that human T-cell leukemia virus type 1 (HTLV-1) infection has the potential to perturb thymic human αβ T-cell development. To verify that inference and to clarify the impact of HTLV-1 infection on human T-cell development, we investigated the in vivo effects of HTLV-1 infection in a “Human Immune System” (HIS) Rag2-/-γc-/- mouse model. These mice were infected with HTLV-1, at a time when the three main subpopulations of human thymocytes have been detected. In all but two inoculated mice, the HTLV-1 provirus was found integrated in thymocytes; the proviral load increased with the length of the infection period. In the HTLV-1-infected mice we observed alterations in human T-cell development, the extent of which correlated with the proviral load. Thus, in the thymus of HTLV-1-infected HIS Rag2-/-γc-/- mice, mature single-positive (SP) CD4+ and CD8+ cells were most numerous, at the expense of immature and double-positive (DP) thymocytes. These SP cells also accumulated in the spleen. Human lymphocytes from thymus and spleen were activated, as shown by the expression of CD25: this activation was correlated with the presence of tax mRNA and with increased expression of NF-kB dependent genes such as bfl-1, an anti-apoptotic gene, in thymocytes. Finally, hepato-splenomegaly, lymphadenopathy and lymphoma/thymoma, in which Tax was detected, were observed in HTLV-1-infected mice, several months after HTLV-1 infection. These results demonstrate the potential of the HIS Rag2-/-γc-/- animal model to elucidate the initial steps of the leukemogenic process induced by HTLV-1.

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<![CDATA[Virus-Encoded microRNAs: An Overview and a Look to the Future]]> https://www.researchpad.co/article/5989da70ab0ee8fa60b94b79

MicroRNAs (miRNAs) are small RNAs that play important roles in the regulation of gene expression. First described as posttranscriptional gene regulators in eukaryotic hosts, virus-encoded miRNAs were later uncovered. It is now apparent that diverse virus families, most with DNA genomes, but at least some with RNA genomes, encode miRNAs. While deciphering the functions of viral miRNAs has lagged behind their discovery, recent functional studies are bringing into focus these roles. Some of the best characterized viral miRNA functions include subtle roles in prolonging the longevity of infected cells, evading the immune response, and regulating the switch to lytic infection. Notably, all of these functions are particularly important during persistent infections. Furthermore, an emerging view of viral miRNAs suggests two distinct groups exist. In the first group, viral miRNAs mimic host miRNAs and take advantage of conserved networks of host miRNA target sites. In the larger second group, viral miRNAs do not share common target sites conserved for host miRNAs, and it remains unclear what fraction of these targeted transcripts are beneficial to the virus. Recent insights from multiple virus families have revealed new ways of interacting with the host miRNA machinery including noncanonical miRNA biogenesis and new mechanisms of posttranscriptional cis gene regulation. Exciting challenges await the field, including determining the most relevant miRNA targets and parlaying our current understanding of viral miRNAs into new therapeutic strategies. To accomplish these goals and to better grasp miRNA function, new in vivo models that recapitulate persistent infections associated with viral pathogens are required.

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<![CDATA[Molecular and Cellular Mechanisms of KSHV Oncogenesis of Kaposi's Sarcoma Associated with HIV/AIDS]]> https://www.researchpad.co/article/5989da0fab0ee8fa60b79271 ]]> <![CDATA[Methylated DNA Recognition during the Reversal of Epigenetic Silencing Is Regulated by Cysteine and Serine Residues in the Epstein-Barr Virus Lytic Switch Protein]]> https://www.researchpad.co/article/5989db28ab0ee8fa60bd0bfc

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma. Like all herpesviruses, the EBV life cycle alternates between latency and lytic replication. During latency, the viral genome is largely silenced by host-driven methylation of CpG motifs and, in the switch to the lytic cycle, this epigenetic silencing is overturned. A key event is the activation of the viral BRLF1 gene by the immediate-early protein Zta. Zta is a bZIP transcription factor that preferentially binds to specific response elements (ZREs) in the BRLF1 promoter (Rp) when these elements are methylated. Zta's ability to trigger lytic cycle activation is severely compromised when a cysteine residue in its bZIP domain is mutated to serine (C189S), but the molecular basis for this effect is unknown. Here we show that the C189S mutant is defective for activating Rp in a Burkitt's lymphoma cell line. The mutant is compromised both in vitro and in vivo for binding two methylated ZREs in Rp (ZRE2 and ZRE3), although the effect is striking only for ZRE3. Molecular modeling of Zta bound to methylated ZRE3, together with biochemical data, indicate that C189 directly contacts one of the two methyl cytosines within a specific CpG motif. The motif's second methyl cytosine (on the complementary DNA strand) is predicted to contact S186, a residue known to regulate methyl-ZRE recognition. Our results suggest that C189 regulates the enhanced interaction of Zta with methylated DNA in overturning the epigenetic control of viral latency. As C189 is conserved in many bZIP proteins, the selectivity of Zta for methylated DNA may be a paradigm for a more general phenomenon.

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<![CDATA[Cyclosporine Inhibits a Direct Interaction between Cyclophilins and Hepatitis C NS5A]]> https://www.researchpad.co/article/5989dacfab0ee8fa60bb57ed

Background

Hepatitis C Virus (HCV) infection is a leading indication for liver transplantation. HCV infection reoccurs almost universally post transplant, decreasing both graft longevity and patient survival. The immunosuppressant, cyclosporine A (CsA) has potent anti-HCV activity towards both HCV replicons and the genotype 2a cell culture infectious virus. Previously, we isolated mutations in the 1bN replicon with less sensitivity to CsA that mapped to both NS5A and NS5B regions of the virus. Mutations in NS5A alone conferred decreased CsA susceptibility regardless of NS5B mutations.

Methodology/Principal Findings

We examined the mechanisms by which NS5A mutations contribute to CsA resistance and if they are strain dependent. Using in vitro mutagenesis, the amino acid position 321 mutation of NS5A was restored to the wild-type tyrosine residue conferring partial CsA susceptibility on the mutant replicon. The 321 mutation also alters CsA susceptibility of the JFH cell culture virus. Additionally, we demonstrated a novel CsA-sensitive interaction between NS5A and both cyclophilin A and B. Both the mutant NS5A and wild type NS5A bind cyclophilin in vitro. The NS5A: cyclophilin interaction requires both the NS5A region identified by the resistance mutants and cyclophilin catalytic residues. In cell culture, NS5A from CsA resistant mutant has an enhanced interaction with cyclophilin B. Additionally; NS5B facilitates a stronger binding of mutant NS5A to endogenous cyclophilin B than wild-type in cell culture.

Conclusions/Significance

Collectively, this data suggests direct interactions between cyclophilins and NS5A are critical to understand for optimal use of cyclophilin inhibitors in anti-HCV therapy.

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