No description available.

Human cytomegalovirus (HCMV) coexists indefinitely in infected individuals through a poorly characterized latent infection in hematopoietic cells. We previously demonstrated a requirement for UL138 in promoting a latent infection in CD34+ hematopoietic progenitor cells (HPCs). UL138 is encoded on three co-terminal transcripts of, 1.7-, 2.7-, and 3.6-kilobases. Interestingly, the UL138 protein product (pUL138) is necessary but insufficient for HCMV latency. The mechanisms by which pUL138 contributes to the latent infection are unknown, however other viral determinants are required for the latent infection. We identified 3 novel proteins pUL133, pUL135, and pUL136 encoded on the UL138 transcripts. Similar to pUL138, pUL133, pUL135, and pUL136 are Golgi localized type I transmembrane proteins expressed with early kinetics during productive infection. We have named these UL138 related proteins, CLAMPs for HCMV Latency Associated Membrane Proteins. Through a systematic immunoprecipitation analysis, we identified interactions between the CLAMPs and characterized an interaction between pUL133 and pUL138. Further, we mapped the interacting region to a specific domain in the C-terminal, cytosolic tail of pUL138. Additionally, we show that each of the CLAMPs has the ability to self-associate. The localization of the CLAMPs to the Golgi suggests that these proteins likely promote HCMV latency through a novel mechanism involving Golgi functions. Additionally, through a Y2H screen of a human bone marrow cDNA library, we identified an interaction between pUL138 and the heat shock protein 40 (Hsp40) variant MRJ. We confirmed this interaction in mammalian cells and mapped the pUL138 region responsible for this interaction to a domain in the cytoplasmic tail of pUL138. We also demonstrated additional MRJ interactions with pUL133 and pUL136. Importantly, pUL138 specifically interacts with Hsp40 variants during productive infection. Preliminary data suggest that HCMV infection up regulates MRJ mRNA expression and recombinant viruses lacking pUL138 show a disproportionate up regulation of MRJ. pUL138 is the first HCMV protein demonstrated to promote a latent infection. While the mechanisms by which pUL138 contributes to latency remain unknown, the interaction with other CLAMPs and with MRJ, suggest that pUL138 may cooperate with other CLAMPs to modulate the cellular stress response at the Golgi to promote HCMV latency.

miRNAs are a species of small-regulatory RNA that post-transcriptionally regulate gene expression via the RNA induced silencing complex (RISC). They are encoded ubiquitously among animals and plants, and have recently been shown to be encoded by the majority of herpesviruses. It seems likely that herpesvirus encoded miRNAs have evolved as a tool for the manipulation of host-cellular and viral-gene expression during infection. Human cytomegalovirus (HCMV) is a clinically important herpesvirus that represents a significant cause of morbidity and mortality in the immune-compromised. HCMV encodes as many as 25 miRNAs during infection, but the function of the majority of these is not known. Identifying the targets of HCMV miRNAs will not only establish a basis for understanding the role of miRNAs within the context of HCMV infection, but also provide a means for discovering novel host-virus interactions. Using RISC immunoprecipitation and siRNA screening, host-cellular targets of viral miRNAs that play important roles in the biology of HCMV were identified. ATP6VOC, a key component of the vacuolar-ATPase, was shown to be a target of miR-US25-1 and subsequent siRNA knockdown of ATP6VOC resulted in the almost complete inhibition of infectious virion production. Despite this, ATP6VOC knock-down did not inhibit viral entry, DNA synthesis, or gene expression, highlighting a possible role for ATP6VOC in the assembly and egress of HCMV. A critical step in HCMV assembly and egress is the formation of the juxta-nuclear virion assembly compartment (VAC). The HCMV VAC is derived from host-cellular endocytic and secretory vacuoles, and is crucial for the efficient nuclear egress of nucleocapsids, cyotplasmic tegumentation, final envelopment, and the egress of mature virions. Using siRNA knock-down, immunofluorescence-microscopy, and western-blot analysis, a crucial role for ATP6VOC and v-ATPase function in the formation of the VAC was demonstrated. siRNA knock-down of ATP6VOC resulted in a failure in the reorganisation of trans-golgi and early-endosomal compartments during infection, resulting in a failure in VAC formation. These findings demonstrate a crucial role for ATP6VOC during infection, and in so doing identify a novel host factor that is required for HCMV assembly.

Mechanisms involved in the establishment of HCMV latency are poorly understood, however, work in our laboratory has demonstrated the ULb' encoded protein, pUL138, as the first viral determinant to function in the establishment of HCMV latency in CD34+ hematopoietic progenitor cells (HPCs). This work characterizes the transcripts that encode pUL138, identifies three novel ULb' proteins (pUL133, pUL135, and pUL136) and represents the first demonstration of an internal ribosome entry site (IRES) mediated expression of pUL138. pUL138 is encoded on three polycistronic transcripts of 3.6-, 2.7- and 1.4-kb in length. pUL133, pUL135 and truncated pUL136, are expressed on the 3.6-, 2.7- and 1.4-kb transcripts, respectively, in addition to pUL138. We demonstrate that pUL138 expression is inducible from the IRES on the 3.6- and 2.7-kb transcripts under conditions of cellular stress, whereas pUL138 expression from the 1.4-kb transcript is inhibited under these same conditions. Differential utilization of the UL138 transcripts and their respective encoded proteins may regulate the outcome of viral infection in a cell type or cell context dependent manner. The interaction of these proteins during HCMV latency is the focus of ongoing research. In addition, this work represents preliminary data regarding the type I interferon (IFN) response during HCMV during productive infection in MRC5 fibroblasts and during the establishment of HCMV latency in CD34+ HPCs.

No description available.

HCMV is a common viral pathogen that infects most of the world's population by early adulthood. It is typically asymptomatic in immunologically healthy individuals but causes severe disease in immunocompromised patients and congenitally infected infants. HCMV glycoproteins are highly polymorphic, and various types of associations have been suggested between glycoprotein types and the pathogenicity of the virus. Several studies on viruses other than HCMV have related the glycosylation of the viral glycoproteins to virulence. This project aimed to determine whether there is a robust relationship between the individual glycoprotein sequence and its glycosylation, how this influences the growth characteristic of the virus and whether this is related to its pathogenicity. Glycosylation patterns of 89 clinical specimens of different infection categories and specimen types were correlated with genetic sequence alterations of the virus glycoproteins (gB, gH, gL, gM, gN, gO), followed by determining whether mutation results in specific changes in glycosylation. The aim was approached using a cell culture model and a quantitative lectin-based assay (ELLA). A significantly increased glycosylation level for the following genotypes: mixed gH, gN4a, gO4, mixed gL was detected. Whereas a decreased pattern was found to be associated with gH1, gH2, gN3a, gO1a and gL2 genotypes (P < 0.05). Glycoproteins of strains isolated from respiratory specimens were significantly highly glycosylated compared to the blood and urine samples, and from blood specimens compared to the urine samples (P < 0.05). Furthermore, strains from congenitally infected infants and urine samples had a significantly higher growth rate than others tested. No direct association between the virus growth and its virulence was found. These findings demonstrate that glycosylation of glycoproteins in HCMV is affected by the glycoprotein polymorphisms and signifies a potentially important mechanism for avoidance of antibody-mediated neutralization, which, in turn, facilitates HCMV pathogenicity. This phenomenon requires further study and may have application for the selection of novel targets for diagnosis, vaccine development and other preventive measures to combat diseases caused by this virus.

Orientador: Maria Cristina Carlan da Silva / Dissertação (mestrado) - Universidade Federal do ABC. Programa de Pós-Graduação em Biossistemas, 2012

Le traitement de cellules par des aminobisphosphonates (ABP) induit une accumulation intracellulaire de molécules activatrices des lymphocytes T Vy9Vd2 (IPP, ApppI). Alors que ces lymphocytes ne semblent pas naturellement activés lors d'une infection par le cytomégalovirus humain (HCMV), le laboratoire a mis en évidence l'existence d'une action synergique entre le HCMV et les ABP sur leur activation. Ma thèse a eu pour objectif d'analyser le mécanisme de cette synergie et d'évaluer le potentiel immuno-thérapeutique des ABP dans le cadre d'une infection HCMV. Nous avons montré qu'après sensibilisation par des ABP in vitro, des cellules infectées sont fortement activatrices des T Vy9Vd2. Les fibroblastes traités par des ABP activent la production d'IFN-y par les T Vy9Vd2 mais pas la production de TNF. L'infection de ces fibroblastes par le HCMV induit une augmentation de la production d'IFN-y et stimule la production de TNF par des T Vy9Vd2. Cette activation a été observée avec des lymphocytes T Vy9Vd2 établis en lignées cellulaires mais aussi avec des cellules Vy9Vd2 isolées directement de sang périphérique. De plus, cette augmentation de production de cytokines est observée avec différentes souches virales (souche de laboratoire et isolats cliniques) et différents types cellulaires permissifs pour HCMV. Nous avons également montré que l'infection par le HCMV entraine une surproduction d'IPP et d'ApppI dans les cellules cibles traitées aux ABP, ce qui explique en partie l'augmentation de la sécrétion de cytokines par les T Vy9Vd2. Enfin, nous avons mis en évidence que ces T Vy9Vd2 sont capables de limiter la réplication et la production virale suite à un traitement par des ABP, tout en préservant les cellules non infectées. Selon nos études, cette activité antivirale implique la production des cytokines IFN-y et TNF et non l'activité cytotoxique des T Vy9Vd2. Par conséquent, mes travaux de thèse fournissent une preuve de concept pour une application thérapeutique des ABP dans le cadre d'une infection par le HCMV. / Aminobisphosphonates (ABP) treatment of cells induces intracellular accumulation of molecules (IPP, ApppI) which stimulate human Vy9Vd2 T cells. Although these lymphocytes do not appear naturally activated during human cytomegalovirus (HCMV) infection, the laboratory demonstrated a synergistic effect of HCMV and ABP on Vy9Vd2 T cell activation. My PhD thesis aimed to analyze the mechanism of this synergy and evaluate the immunotherapeutic potential of ABP in the context of HCMV infection. After ABP treatment of cells in vitro, we showed that HCMV-infected cells strongly activated Vy9Vd2 T cells. ABP-treated fibroblasts activate Vy9Vd2 T cells to produce IFN-y but not TNF. The HCMV infection of these fibroblasts stimulates TNF secretion and an increased production of IFN-y, indicating that Vy9Vd2 cells can sense HCMV infection. Increased cytokine production was observed with Vy9Vd2 T cell lines and "fresh" Vy9Vd2 directly isolated of blood. Moreover, Vy9Vd2 T cell activation was observed with most HCMV strains (laboratory strains or clinical isolates) and different HCMV-permissive cells. We also showed that HCMV infection induces an overproduction of IPP and ApppI in ABP-treated cells, which explains in part the increased cytokine production by Vy9Vd2 T cells. At last, we demonstrated the capacity of Vy9Vd2 T cells to limit viral replication and production after ABP treatment while preserving uninfected cells. Our experiments indicate that this antiviral activity involves IFN-y and TNF secretion by Vy9Vd2 T cells but not their cytotoxicity activity. Consequently, my work provides a proof of concept of the therapeutic potential of ABP in the context of HCMV infection.

Human cytomegalovirus (CMV) is a significant infectious agent causing disease in immunocompromised HIV-infected patients, transplant recipients, and neonates. The current antiviral therapeutic strategy against CMV is limited in its utility due to the inherent toxicity and lack of bioavailability of currently available anti-CMV agents, ganciclovir (GCV), cidofovir (CDV), and foscarnet (FOS). The development of the prodrug of GCV, valganciclovir (val-GCV), has vastly improved the bioavailability profile of GCV. However, val-GCV demonstrates limited effectiveness against tissue-invasive CMV diseases as side effects involved with traditional intravenously administered GCV such as haematologic and reproductive toxicities remain. In addition, the emergence of antiviral resistant CMV mutant strains due to prolonged treatment with currently available antivirals necessitates the development of novel anti-CMV agents with reduced toxicity and improved bioavailability. In this study, select groups of novel compounds were analysed for their potential for further development as anti-CMV agents. Three groups of compounds were identified based on two screening methods which included the computer simulated screening process of compounds known as in silico screening and the traditional method of random screening. The first group of compounds (CATi) were identified by in silico screening against the CMV DNA polymerase catalytic aspartate triad, resulting in the identification of 31 compounds with the potential for inhibitory activity against CMV. The second group of compounds (PRO-i) were identified through in silico screening against the CMV protease, identifying a total of 18 lead compounds exhibiting structural complementarity with CMV protease. The third and final group of compounds (TPEX) were identified through random screening and consisted of plant extracts purified from tropical plants. All three compounds were initially screened for cytotoxicity against human fibroblasts. Plaque reduction assays were performed using compounds with acceptable levels of cytotoxicity to determine the ability of the compounds to inhibit the replication of the laboratory antiviral sensitive CMV strain, Towne. Two of the PRO-i compounds demonstrated good antiviral activity against CMV. Eleven percent (2/18) of the PRO-i compounds inhibited CMV replication, with PRO-i-43 and PRO-i??-44 displaying mean 50% inhibitory concentrations (IC50) of 4.8 ?? 1.2 ??M and 8.04 ??M, respectively. PRO-i-43 and PRO-i-44 are thus good candidates for further development as novel antiviral agents against CMV. The majority of CATi and TPEX compounds displayed significant cytotoxicity against human fibroblasts and compounds with acceptable levels of cytotoxicities did not significantly inhibit CMV replication. However, the identification of compounds with low cytotoxicities provides a good foundation for further development of novel anti-CMV agents with superior antiviral activity. In silico screening against three-dimensional viral protein models is a useful strategy for the identification of novel antiviral agents with the potential for inhibitory activity against CMV. Structural modification to produce potent derivatives of the identified anti-CMV compounds (PRO-i-43 and PRO-i-44) is a good option for the further development of novel antiviral agents against CMV. Such further examination of the identified compounds with anti-CMV activity is required to investigate their activity against not only antiviral sensitive CMV strains but also resistant CMV strains. Further investigations will yield new insights into their target, allowing further identification of compounds with potential anti-CMV activity with pharmaceutical application.

The human cytomegalovirus (CMV) is one of the viruses which extensively infect in the world. In order to grow and reproduce, the CMV invades designated cellular lives and influences their behavior. The origin of replication (also called the replication origin) is a particular sequence in the CMV DNA genome at which replication is initiated. In this study, we develop some statistical tests of complementary palindromes, which can be applied to narrow the search for replication origin of the CMV DNA sequence. Let X_(2k) be the number of complementary palindromes with length 2k and Y_(2k) be the number of non-covered complementary palindromes with length 2k inside a given DNA sequence. Consider the null hypothesis that the marginal probabilities of the four nucleotides remain the same (1/4) over the given sequence versus the alternative hypothesis that the marginal probabilities are different. The likelihood ratio test based on the joint distributions of Y_(18) and Y_(2k) | (Y_(2(k+1)), ...,Y_(18)), where k=1, ..., 8, under the null and the alternative hypotheses are derived. The null distribution of the test statistic is approximated by a scaled chi-squared distribution. The scale parameter and the degree of freedom are estimated by the method of moments. The Pearson's chi-squared test based on the marginal distributions of X_(2k), where k=1, ..., 9. The null distribution of the test statistic is also approximated by a scaled chi-squared distribution. There is an another focus about ratios statistics X_(2k)/X_(2(k+1)) and Y_(2k)/Y_(2(k+1)), which approximate a specific value under the null hypotheses. Simulation studies are performed to confirm the theoretical findings.