measuring-granulocyte-monocyte-phagocytosis-oxidative-burst-activity

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

David H. Murdock Research Institute

Human herpesvirus-8 (HHV-8) is implicated in the pathogenesis of Kaposi's sarcoma. HHV-8 envelope glycoprotein B possesses the RGD motif known to interact with integrin molecules, and HHV-8 infectivity was inhibited by RGD peptides, antibodies against RGD-dependent alpha3 and beta1 integrins, and by soluble alpha3beta1 integrin. Expression of human alpha3 integrin increased the infectivity of virus for Chinese hamster ovary cells. Anti-gB antibodies immunoprecipitated the virus-alpha3 and -beta1 complexes, and virus binding studies suggest a role for alpha3beta1 in HHV-8 entry. Further, HHV-8 infection induced the integrin-mediated activation of focal adhesion kinase (FAK). These findings implicate a role for alpha3beta1 integrin and the associated signaling pathways in HHV-8 entry into the target cells.

Integrin alpha3beta1 (CD 49c/29) is a cellular receptor for Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) entry into the target cells.

Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus, implicated in the pathogenesis of Kaposi's sarcoma, utilizes heparan sulfate-like molecules to bind the target cells via its envelope-associated glycoproteins gB and gpK8.1A. HHV-8-gB possesses the Arg-Gly-Asp (RGD) motif, the minimal peptide region of many proteins known to interact with subsets of host cell surface integrins. HHV-8 utilizes alpha3beta1 integrin as one of the receptors for its entry into the target cells via its gB interaction and induces the activation of focal adhesion kinase (FAK) (S. M. Akula, N. P. Pramod, F.-Z. Wang, and B. Chandran, Cell 108:407-419, 2002). Since FAK activation is the first step in the outside-in signaling necessary for integrin-mediated cytoskeletal rearrangements, cell adhesions, motility, and proliferation, the ability of HHV-8-gB to mediate the target cell adhesion was examined. A truncated form of gB without the transmembrane and carboxyl domains (gBdeltaTM) and a gBdeltaTM mutant (gBdeltaTM-RGA) with a single amino acid mutation (RGD to RGA) were expressed in a baculovirus system and purified. Radiolabeled HHV-8-gBdeltaTM, gBdeltaTM-RGA, and deltaTMgpK8.1A proteins bound to the human foreskin fibroblasts (HFFs), human dermal microvascular endothelial (HMVEC-d) cells, human B (BJAB) cells, and Chinese hamster ovary (CHO-K1) cells with equal efficiency, which was blocked by preincubation of proteins with soluble heparin. Maxisorp plate-bound gBdeltaTM protein induced the adhesion of HFFs and HMVEC-d and monkey kidney epithelial (CV-1) cells in a dose-dependent manner. In contrast, the gBdeltaTM-RGA and DeltaTMgpK8.1A proteins did not mediate adhesion. Adhesion mediated by gBdeltaTM was blocked by the preincubation of target cells with RGD-containing peptides or by the preincubation of plate-bound gBdeltaTM protein with rabbit antibodies against gB peptide containing the RGD sequence. In contrast, adhesion was not blocked by the preincubation of plate-bound gBdeltaTM protein with heparin, suggesting that the adhesion is mediated by the RGD amino acids of gB, which is independent of the heparin-binding domain of gB. Integrin-ligand interaction is dependent on divalent cations. Adhesion induced by the gBdeltaTM was blocked by EDTA, thus suggesting the role of integrins in the observed adhesions. Focal adhesion components such as FAK and paxillin were activated by the binding of gBdeltaTM protein to the target cells but not by gBdeltaTM-RGA protein binding. Inhibition of FAK phosphorylation by genistein blocked gBdeltaTM-induced FAK activation and cell adhesion. These findings suggest that HHV-8-gB could mediate cell adhesion via its RGD motif interaction with the cell surface integrin molecules and indicate the induction of cellular signaling pathways, which may play roles in the infection of target cells and in Kaposi's sarcoma pathogenesis.

Human herpesvirus 8 envelope glycoprotein B mediates cell adhesion via its RGD sequence.

Kaposi's sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (HHV-8) DNA and transcripts have been detected in the B cells, macrophages, keratinocytes, and endothelial and epithelial cells of KS patients. In vitro, HHV-8 infects human B, endothelial, epithelial, and fibroblast cells, as well as animal cells, and the infection is characterized by (i) absence of lytic replication by the input virus and (ii) latent infection. For its initial binding to target cells, HHV-8 uses ubiquitous heparan sulfate molecules via its envelope-associated glycoproteins gB and gpK8.1A. HHV-8 also interacts with the alpha3beta1 integrin via its glycoprotein gB, and virus binding studies suggest that alpha3beta1 is one of the HHV-8 entry receptors (S. M. Akula, N. P. Pramod, F. Z. Wang, and B. Chandran, Cell 108:407-419, 2002). In this study, morphological and biochemical techniques were used to examine the entry of HHV-8 into human foreskin fibroblasts (HFF). HHV-8 was detected in coated vesicles and in large, smooth-surfaced endocytic vesicles. Fusion of viral envelope with the vesicle wall was also observed. In immune electron microscopy, anti-HHV-8 gB antibodies colocalized with virus-containing endocytic vesicles. In fluorescence microscopic analyses, transferrin was colocalized with HHV-8. HHV-8 infection was significantly inhibited by preincubation of cells with chlorpromazine HCl, which blocks endocytosis via clathrin-coated pits, but not by nystatin and cholera toxin B, which blocks endocytosis via caveolae and induces the dissociation of lipid rafts, respectively. Infection was also inhibited by blocking the acidification of endosomes by NH(4)Cl and bafilomycin A. Inhibition of HHV-8 open reading frame 73 gene expression by chlorpromazine HCl, bafilomycin A, and NH(4)Cl demonstrated that the virions in the vesicles could proceed to cause an infection. Taken together, these findings suggest that for its infectious entry into HFF, HHV-8 uses clathrin-mediated endocytosis and a low-pH intracellular environment.

Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) infection of human fibroblast cells occurs through endocytosis.

The human cytomegalovirus (HCMV) US12 gene family is a group of predicted seven-transmembrane, G-protein-coupled receptor-related proteins, about which little is known. Specific rabbit polyclonal antibodies detected US17 and US18 beginning 54 and 36 h after infection, respectively, with expression of both proteins dependent on viral DNA synthesis. While US14 and US18 are expressed exclusively in the cytoplasm, we unexpectedly found abundant expression of US17 in both the cytoplasm and nucleoplasm. N- and C-terminally tagged versions of US17 were readily detected in the cytoplasm of transfected mammalian cells, but not in nuclei, suggesting that nuclear localization involves other viral proteins or an infection-triggered cellular process. There was no specific colocalization between US17 and other nuclear expressed HCMV-encoded proteins (IE-2, DNA polymerase processivity factor, and pp28/UL99). To determine whether the observed nuclear localization might be the product of a process by which a soluble C-terminal segment of the full-length protein is expressed, we constructed a recombinant virus that incorporates a synthetic epitope at its N terminus, which in conjunction with the antipeptide antibody that targets its predicted cytoplasmic C-terminal segment, enables simultaneous independent detection of both termini. In cells infected with the recombinant, the US17 N and C termini had limited colocalization, with the N-terminal segment not detected in nuclei, supporting the segmentation hypothesis. Consistent with this, a fragment with an apparent molecular size of 10 kDa was detected by immunoblotting. We have identified the first viral example of a seven-transmembrane protein that is either segmented or expressed in nuclei. Further study will be required to learn the mechanism by which this occurs and the function of the nuclear localizing segment. This likely represents yet another mechanism by which a virus has hijacked or modified cellular regulatory pathways for its benefit.

Infection-dependent nuclear localization of US17, a member of the US12 family of human cytomegalovirus-encoded seven-transmembrane proteins.

Human cytomegalovirus (HCMV; Human herpesvirus 5) and the other betaherpesviruses encode a number of distinct gene families, including the US12 family, which is represented only in the cytomegaloviruses of higher primates, and is comprised of a set of 10 contiguous genes (US12 through US21), each encoding a seven-transmembrane (7TM) protein. Nonessential for replication in cell culture but well-conserved among clinical isolates, little is known of possible US12 family member functions, other than a previously identified amino acid sequence similarity between US21 and a group of 7TM proteins that include known inhibitors of apoptosis, and a very limited description of similarity between US12 family members and G-protein-coupled receptors (GPCR). As a prelude to biochemical analysis, we have conducted a detailed analysis of the relationships among US12 family members and between these proteins and other proteins, particularly GPCR and other 7TM molecules. In most cases, the closest relatives of individual genes are their colinear counterparts in the other viruses. Thus, the initial duplication and divergence events that resulted in the current version of the US12 family preceded divergence of the rhesus and hominoid lineages. Our phylogenetic analysis indicates that the US12 family represents a distinct branch of the 7TM superfamily. Although they are distantly related, at least some of the US12 family members may have GPCR-related properties, but they are also likely to embody functions and mechanisms that differ from more conventional GPCRs. Our analyses suggest that the 7TM structure of US12 family members constitutes a functionally flexible structural scaffold that can be readily adapted to diverse functional ends. This strategy may be the driving force in the emergence of the several families of duplicated and diverged betaherpesvirus genes.

Primate cytomegalovirus US12 gene family: a distinct and diverse clade of seven-transmembrane proteins.

We conducted a cross-sectional study of beta-herpesviruses in febrile pediatric oncology patients (n = 30), with a reference group of febrile pediatric solid-organ transplant recipients (n = 9). One (3.3%) of 30 cancer patients and 3 (33%) of 9 organ recipients were PCR positive for cytomegalovirus. Four (13%) of 30 cancer patients and 3 (33%) of 9 transplant recipients had human herpesvirus 6B (HHV-6B) DNAemia, which was more common within 6 months of initiation of immune suppression (4 of 16 vs. 0 of 14 cancer patients; p = 0.050). HHV-6A and HHV-7 were not detected. No other cause was identified in children with HHV-6B or cytomegalovirus DNAemia. One HHV-6B-positive cancer patient had febrile disease with concomitant hepatitis. Other HHV-6B-positive children had mild "viral" illnesses, as did a child with primary cytomegalovirus infection. Cytomegalovirus and HHV-6B should be included in the differential diagnosis of febrile disease in children with cancer.

Beta-herpesviruses in febrile children with cancer.

The human genome encodes over 500 microRNAs (miRNAs), small RNAs (19 to 26 nucleotides [nt]) that regulate the expressions of diverse cellular genes. Many cellular processes are altered through a variety of mechanisms by human cytomegalovirus (HCMV) infection. We asked whether HCMV infection leads to changes in the expression of cellular miRNAs and whether HCMV-regulated miRNAs are important for HCMV replication. Levels of most miRNAs did not change markedly during infection, but some were positively or negatively regulated. Patterns of miRNA expression were linked to the time course of infection. Some similarly reregulated miRNAs share identical or similar seed sequences, suggesting coordinated regulation of miRNA species that have shared targets. miRNAs miR-100 and miR-101 were chosen for further analyses based on their reproducible changes in expression after infection and on the basis of having predicted targets in the 3' untranslated regions (3'-UTR) of genes encoding components of the mammalian target of rapamycin (mTOR) pathway, which is important during HCMV infection. Reporter genes that contain the 3'-UTR of mTOR (predicted targets for miR-100 and miR-101) or raptor (a component of the mTOR pathway; predicted site for miR-100) were constructed. Mimics of miR-100 and miR-101 inhibited expression from the mTOR construct, while only miR-100 inhibited the raptor construct. Together, miR-100 and miR-101 reduced mTOR protein levels. While the miR-100 and miR-101 mimics individually modestly inhibited production of infectious progeny, much greater inhibition was achieved with a combination of both (33-fold). Our key finding is that HCMV selectively manipulates the expression of some cellular miRNAs to help its own replication.

Human cytomegalovirus infection alters the expression of cellular microRNA species that affect its replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface heparan sulfate (HS) and alpha3beta1 integrin during the early stages of infection of human dermal microvascular endothelial cells (HMVEC-d) and human foreskin fibroblasts (HFF), and these interactions are followed by virus entry overlapping with the induction of preexisting host cell signal pathways. KSHV also utilizes the amino acid transporter protein xCT for infection of adherent cells, and the xCT molecule is part of the cell surface heterodimeric membrane glycoprotein CD98 (4F2 antigen) complex known to interact with alpha3beta1 and alphaVbeta3 integrins. KSHV gB mediates adhesion of HMVEC-d, CV-1, and HT-1080 cells and HFF via its RGD sequence. Anti-alphaV and -beta1 integrin antibodies inhibited the cell adhesion mediated by KSHV-gB. Variable levels of neutralization of HMVEC-d and HFF infection were observed with antibodies against alphaVbeta3 and alphaVbeta5 integrins. Similarly, variable levels of inhibition of virus entry into adherent HMVEC-d, 293 and Vero cells, and HFF was observed by preincubating virus with soluble alpha3beta1, alphaVbeta3, and alphaVbeta5 integrins, and cumulative inhibition was observed with a combination of integrins. We were unable to infect HT1080 cells. Virus binding and DNA internalization studies suggest that alphaVbeta3 and alphaVbeta5 integrins also play roles in KSHV entry. We observed time-dependent temporal KSHV interactions with HMVEC-d integrins and CD98/xCT with three different patterns of association and dissociation. Integrin alphaVbeta5 interaction with CD98/xCT predominantly occurred by 1 min postinfection (p.i.) and dissociated at 10 min p.i., whereas alpha3beta1-CD98/xCT interaction was maximal at 10 min p.i. and dissociated at 30 min p.i., and alphaVbeta3-CD98/xCT interaction was maximal at 10 min p.i. and remained at the observed 30 min p.i. Fluorescence microscopy also showed a similar time-dependent interaction of alphaVbeta5-CD98. Confocal-microscopy studies confirmed the association of CD98/xCT with alpha3beta1 and KSHV. Preincubation of KSHV with soluble heparin and alpha3beta1 significantly inhibited this association, suggesting that the first contact with HS and integrin is an essential element in subsequent CD98-xCT interactions. Anti-CD98 and xCT antibodies did not block virus binding and entry and nuclear delivery of viral DNA; however, viral-gene expression was significantly inhibited, suggesting that CD98-xCT play roles in the post-entry stage of infection, possibly in mediating signal cascades essential for viral-gene expression. Together, these studies suggest that KSHV interacts with functionally related integrins (alphaVbeta3, alpha3beta1, and alphaVbeta5) and CD98/xCT molecules in a temporal fashion to form a multimolecular complex during the early stages of endothelial cell infection, probably mediating multiple roles in entry, signal transduction, and viral-gene expression.

Kaposi's sarcoma-associated herpesvirus forms a multimolecular complex of integrins (alphaVbeta5, alphaVbeta3, and alpha3beta1) and CD98-xCT during infection of human dermal microvascular endothelial cells, and CD98-xCT is essential for the postentry stage of infection.

&#x867D;&#x7136;&#x8BB8;&#x591A;&#x836F;&#x7269;&#x5728;&#x7EC6;&#x80DE;&#x57F9;&#x517B;&#x7CFB;&#x7EDF;&#x4E2D;&#x6291;&#x5236;&#x7684;&#x7231;&#x6CFC;&#x65AF;&#x5766;-&#x5DF4;&#x5C14;&#x75C5;&#x6BD2; (EBV) &#x7684;&#x590D;&#x5236;&#xFF0C;&#x4F46;&#x4ECD;&#x6709;&#x4E0D;&#x662F;&#x6709;&#x6548;&#x7684;&#x548C;&#x6838;&#x51C6;&#x7528;&#x4E8E;&#x4E3B; EBV &#x611F;&#x67D3;&#x7684;&#x836F;&#x7269;&#x3002;&#x6700;&#x8FD1;&#xFF0C;maribavir (MBV) l ribofuranoside &#x82EF;&#x5E76;&#x54AA;&#x5511;&#xFF0C;&#x5DF2;&#x8BC1;&#x660E;&#x662F;&#x4E00;&#x79CD;&#x5F3A;&#x6548;&#x548C;&#x65E0;&#x6BD2;&#x7684;&#x6291;&#x5236;&#x5242; EBV &#x590D;&#x5236;&#x5E76;&#x6709;&#x622A;&#x7136;&#x6709;&#x522B;&#x7684;&#x65E0;&#x73AF;&#x6838;&#x82F7;&#x7C7B;&#x4F3C;&#x7269;&#x5982;&#x963F;&#x6614;&#x6D1B;&#x97E6; &#xFF08;ACV&#xFF09;&#xFF0C;&#x4E3B;&#x8981;&#x57FA;&#x4E8E; MBV &#x7684;&#x80FD;&#x529B;&#x6765;&#x963B;&#x6B62;&#x76EE;&#x6807;&#x86CB;&#x767D;&#x8D28;&#x7684;&#x78F7;&#x9178;&#x5316;&#x7531; EBV &#x548C;&#x4EBA;&#x7C7B;&#x5DE8;&#x7EC6;&#x80DE;&#x75C5;&#x6BD2;&#x86CB;&#x767D;&#x6FC0;&#x9176;&#x7684;&#x884C;&#x52A8;&#x6A21;&#x5F0F;&#x3002;&#x4F46;&#x662F;&#x7684;&#x6297;&#x75C5;&#x6BD2;&#x836F;&#x7269;&#x7684;&#x673A;&#x5236;&#x662F;&#x590D;&#x6742;&#x7684;&#x56E0;&#x4E3A;&#x6211;&#x4EEC;&#x5DF2;&#x8FDB;&#x884C;&#x5168;&#x9762;&#x5206;&#x6790;&#x4E0E;&#x5B9A;&#x91CF;&#x5B9E;&#x65F6;&#x53CD;&#x5411;&#x8F6C;&#x5F55;&#x7684;&#x805A;&#x5408;&#x9176;&#x94FE;&#x53CD;&#x5E94;-&#x57FA;&#x4E8E;&#x9635;&#x5217;&#x7684;&#x6240;&#x6709; EBV &#x57FA;&#x56E0;&#x7684; RNA &#x8868;&#x8FBE;&#x6C34;&#x5E73;&#x5BF9; MBV &#x7684;&#x5F71;&#x54CD;&#x3002;&#x6211;&#x4EEC;&#x8868;&#x660E;&#xFF0C;&#x5728;&#x5965;&#x5730;&#x5229;&#xFF0C;&#x6BD4;&#x8F83;&#x5B8C;&#x5168;&#x4E0D;&#x540C;&#xFF0C;&#x4EA7;&#x751F;&#x7684;&#x4E24;&#x79CD;&#x836F;&#x7269;&#x7684; RNA &#x8868;&#x8FBE;&#x8C31;&#x662F;&#x4E0E; MBV &#x9020;&#x6210;&#x591A;&#x4E2A;&#x75C5;&#x6BD2;&#x5206;&#x6CCC;&#x660E;&#x663E;&#x7684;&#x6291;&#x5236;&#x548C; ACV &#x5BFC;&#x81F4;&#x51E0;&#x4E4E;&#x6CA1;&#x6709;&#x4EBA;&#x3002;&#x7ED3;&#x679C;&#x5F3A;&#x8C03;&#x4E0D;&#x540C;&#x6A21;&#x5F0F;&#x7684;&#x4E24;&#x79CD;&#x836F;&#x7269;&#x7684;&#x884C;&#x52A8;&#xFF0C;&#x5E76;&#x5EFA;&#x8BAE; MBV &#x7684;&#x884C;&#x52A8;&#x53EF;&#x80FD;&#x4F1A;&#x94FE;&#x63A5;&#x5230;&#x5BF9; EBV &#x901A;&#x8FC7;&#x4E0E;&#x591A;&#x4E2A;&#x75C5;&#x6BD2;&#x86CB;&#x767D;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x7684; BGLF4 &#x57FA;&#x56E0;&#x7684;&#x8F6C;&#x5F55;&#x7684;&#x95F4;&#x63A5;&#x5F71;&#x54CD;&#x3002;

Maribavir &#x6291;&#x5236;&#x75C5;&#x6BD2; DNA &#x590D;&#x5236;&#x9664;&#x4E86;&#x7231;&#x6CFC;&#x65AF;&#x5766;-&#x5DF4;&#x5C14;&#x75C5;&#x6BD2;&#x8F6C;&#x5F55;&#x3002;

Although many drugs inhibit the replication of Epstein-Barr virus (EBV) in cell culture systems, there is still no drug that is effective and approved for use in primary EBV infection. More recently, maribavir (MBV), an l-ribofuranoside benzimidazole, has been shown to be a potent and nontoxic inhibitor of EBV replication and to have a mode of action quite distinct from that of acyclic nucleoside analogs such as acyclovir (ACV) that is based primarily on MBV's ability to block the phosphorylation of target proteins by EBV and human cytomegalovirus protein kinases. However, since the antiviral mechanisms of the drug are complex, we have carried out a comprehensive analysis of the effects of MBV on the RNA expression levels of all EBV genes with a quantitative real-time reverse transcription-PCR-based array. We show that in comparisons with ACV, the RNA expression profiles produced by the two drugs are entirely different, with MBV causing a pronounced inhibition of multiple viral mRNAs and with ACV causing virtually none. The results emphasize the different modes of action of the two drugs and suggest that the action of MBV may be linked to indirect effects on the transcription of EBV genes through the interaction of BGLF4 with multiple viral proteins.

Maribavir inhibits epstein-barr virus transcription in addition to viral DNA replication.

Bien que de nombreux m&#xE9;dicaments inhibent la r&#xE9;plication du virus Epstein-Barr (EBV) dans les syst&#xE8;mes de culture cellulaire, il n&#x26;#39;existe encore aucun m&#xE9;dicament qui soit efficace et approuv&#xE9; pour une utilisation dans la primo-infection EBV. Plus r&#xE9;cemment, maribavir (MBV), un benzimidazole L-ribofuranoside, il a &#xE9;t&#xE9; d&#xE9;montr&#xE9; &#xEA;tre un inhibiteur puissant et non toxique de la r&#xE9;plication EBV et d&#x26;#39;avoir un mode d&#x26;#39;action tout &#xE0; fait distincte de celle des analogues nucl&#xE9;osidiques acycliques tels que l&#x26;#39;acyclovir (ACV) qui est bas&#xE9;e principalement sur la capacit&#xE9; MBV de bloquer la phosphorylation de prot&#xE9;ines cibles par l&#x26;#39;EBV et de l&#x26;#39;homme prot&#xE9;ines kinases cytom&#xE9;galovirus. Toutefois, &#xE9;tant donn&#xE9; que les m&#xE9;canismes antiviraux de la drogue sont complexes, nous avons effectu&#xE9; une analyse exhaustive des effets de MBV sur les niveaux d&#x26;#39;expression d&#x26;#39;ARN de tous les g&#xE8;nes d&#x26;#39;EBV avec une quantitative en temps r&#xE9;el la transcription bas&#xE9;e sur la PCR inverse tableau. Nous montrons que dans les comparaisons avec l&#x26;#39;ACV, les profils d&#x26;#39;expression ARN produites par les deux m&#xE9;dicaments sont tout &#xE0; fait diff&#xE9;rente, avec MBV provoquant une inhibition marqu&#xE9;e de plusieurs ARNm viraux et avec ACV causant pratiquement aucun. Les r&#xE9;sultats mettent en &#xE9;vidence les diff&#xE9;rents modes d&#x26;#39;action des deux m&#xE9;dicaments et sugg&#xE8;rent que l&#x26;#39;action de MBV peut &#xEA;tre li&#xE9;e &#xE0; des effets indirects sur la transcription de g&#xE8;nes EBV par l&#x26;#39;interaction de BGLF4 avec plusieurs prot&#xE9;ines virales.

Maribavir inhibe la transcription du virus d&#x26;#39;Epstein-Barr dans Ajout &#xE0; la r&#xE9;plication d&#x26;#39;ADN viral

Obwohl viele Medikamente die Replikation des Epstein - Barr-Virus (EBV) in Zellkultursysteme hemmen, gibt es noch kein Medikament, das effektive und zugelassen f&uuml;r die Verwendung in prim&auml;re EBV-Infektion ist. Vor kurzem hat Maribavir (MBV), eine l-Ribofuranoside Benzimidazol, gezeigt, ein potenter und ungiftig Inhibitor der EBV-Replikation zu sein, eine Wirkungsweise deutlich von dem der azyklische Nukleosid-Analoga wie Aciclovir (ACV) haben, die in erster Linie auf MBVs-F&auml;higkeit, die Phosphorylierung von Zielproteinen block von EBV und menschlichen Zytomegalovirus Proteinkinasen basiert. Jedoch da die antiviralen Mechanismen der Droge komplex sind, haben wir eine umfassende Analyse der Auswirkungen der MBV auf die RNA Ausdruck alle EBV-Gene mit einem quantitativen Echtzeit reverse Transkription-PCR-basierten Array durchgef&uuml;hrt. Wir zeigen, dass im Vergleich mit ACV, die RNA-Ausdruck-Profile, die zwei Drogen v&ouml;llig unterschiedlich sind, mit MBV verursacht eine ausgepr&auml;gte Hemmung der mehrere virale mRNAs und ACV verursacht praktisch keine. Die Ergebnisse unterstreichen die verschiedenen Wirkungsweisen der beiden Medikamente und schlagen vor, da&szlig; die Aktion des MBV mit indirekten Auswirkungen auf die Transkription von Genen der EBV durch Wechselwirkung von BGLF4 mit mehreren viralen Proteine verbunden sein kann.

Maribavir hemmt Epstein - Barr-Virus-Transkription neben viralen DNA-Replikation.

Anche se molti farmaci di inibire la replicazione del virus di Epstein - Barr (EBV) in sistemi di coltura cellulare, non c&#39;&egrave; ancora nessun farmaco che sia efficace e approvati per l&#39;uso nell&#39;infezione primaria da EBV. Pi&ugrave; recentemente, maribavir (MBV), un benzimidazolo l-ribofuranoside, ha dimostrato di essere un inibitore potente e atossico della replica di EBV e di avere una modalit&agrave; d&#39;azione abbastanza distinta da quella di analoghi nucleosidici aciclici come aciclovir (ACV) che si basa principalmente sulla capacit&agrave; di MBV di bloccare la fosforilazione delle proteine bersaglio da EBV e chinasi citomegalovirus umano. Tuttavia, poich&eacute; i meccanismi antivirali del farmaco sono complessi, abbiamo svolto un&#39;analisi completa degli effetti di MBV sui livelli di espressione di RNA di tutti i geni di EBV con un quantitativa in tempo reale reverse transcription-PCR-based matrice. Che mostriamo nei confronti con ACV, i profili di espressione di RNA prodotti da due farmaci sono completamente diversi, con MBV causando una pronunciata inibizione del mRNA virale pi&ugrave; con ACV provocando praticamente nessuno. I risultati sottolineano le diverse modalit&agrave; di azione dei due farmaci e suggeriscono che l&#39;azione di MBV possa essere collegato agli effetti indiretti sulla trascrizione dei geni di EBV attraverso l&#39;interazione di BGLF4 con pi&ugrave; proteine virali.

Maribavir inibisce la trascrizione del virus epstein - barr oltre alla replicazione del DNA virale.

&#x591A;&#x304F;&#x306E;&#x85AC;&#x5264;&#x306F;&#x3001;&#x7D30;&#x80DE;&#x57F9;&#x990A;&#x7CFB;&#x306B;&#x304A;&#x3051;&#x308B;Epstein-Barr&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#xFF08;EBV&#xFF09;&#x306E;&#x8907;&#x88FD;&#x3092;&#x963B;&#x5BB3;&#x3059;&#x308B;&#x304C;&#x3001;&#x52B9;&#x679C;&#x7684;&#x304B;&#x3064;&#x4E3B;&#x8981;&#x306A;EBV&#x611F;&#x67D3;&#x3067;&#x306E;&#x4F7F;&#x7528;&#x304C;&#x627F;&#x8A8D;&#x3055;&#x308C;&#x306A;&#x3044;&#x85AC;&#x5264;&#x306F;&#x307E;&#x3060;&#x3042;&#x308A;&#x307E;&#x305B;&#x3093;&#x3002;&#x6700;&#x8FD1;&#x3067;&#x306F;&#x3001;maribavir&#xFF08;MBV&#xFF09;&#x3001;L-ribofuranoside&#x30D9;&#x30F3;&#x30BA;&#x30A4;&#x30DF;&#x30C0;&#x30BE;&#x30FC;&#x30EB;&#x3001;&#x3064;&#x307E;&#x308A;EBV&#x8907;&#x88FD;&#x306E;&#x5F37;&#x529B;&#x304B;&#x3064;&#x7121;&#x6BD2;&#x306A;&#x963B;&#x5BB3;&#x5264;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3084;&#x3001;&#x30A2;&#x30B7;&#x30AF;&#x30ED;&#x30D3;&#x30EB;&#xFF08;ACV&#xFF09;&#x306A;&#x3069;&#x306E;&#x975E;&#x74B0;&#x72B6;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30B7;&#x30C9;&#x985E;&#x7E01;&#x4F53;&#x306E;&#x305D;&#x308C;&#x3068;&#x306F;&#x5168;&#x304F;&#x7570;&#x306A;&#x308B;&#x4F5C;&#x7528;&#x6A5F;&#x5E8F;&#x3092;&#x6709;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x4E3B;&#x306B;EBV&#x3068;&#x30D2;&#x30C8;&#x30B5;&#x30A4;&#x30C8;&#x30E1;&#x30AC;&#x30ED;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x30D7;&#x30ED;&#x30C6;&#x30A4;&#x30F3;&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x306B;&#x3088;&#x308B;&#x6A19;&#x7684;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x3092;&#x30D6;&#x30ED;&#x30C3;&#x30AF;&#x3059;&#x308B;MBV&#x306E;&#x80FD;&#x529B;&#x306B;&#x57FA;&#x3065;&#x3044;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x85AC;&#x5264;&#x306E;&#x6297;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x6A5F;&#x69CB;&#x306F;&#x8907;&#x96D1;&#x3067;&#x3042;&#x308A;&#x3001;&#x3057;&#x304B;&#x3057;&#x3001;&#x6211;&#x3005;&#x306F;&#x5B9A;&#x91CF;&#x7684;&#x30EA;&#x30A2;&#x30EB;&#x30BF;&#x30A4;&#x30E0;&#x9006;&#x8EE2;&#x5199;-PCR&#x30D9;&#x30FC;&#x30B9;&#x306E;&#x914D;&#x5217;&#x3092;&#x6301;&#x3064;&#x3059;&#x3079;&#x3066;&#x306E;EBV&#x907A;&#x4F1D;&#x5B50;&#x306E;RNA&#x767A;&#x73FE;&#x30EC;&#x30D9;&#x30EB;&#x306E;MBV&#x306E;&#x5F71;&#x97FF;&#x306E;&#x5305;&#x62EC;&#x7684;&#x306A;&#x5206;&#x6790;&#x3092;&#x884C;&#x3063;&#x305F;&#x3002;&#x6211;&#x3005;&#x306F;&#x3001;MBV&#x306F;&#x3001;&#x8907;&#x6570;&#x306E;&#x30A6;&#x30A4;&#x30EB;&#x30B9;mRNA&#x306E;&#x9855;&#x8457;&#x306A;&#x963B;&#x5BB3;&#x3092;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3059;&#x3068;&#x3057;&#x3001;ACV&#x306F;&#x5B9F;&#x8CEA;&#x7684;&#x306B;&#x3069;&#x308C;&#x3092;&#x8D77;&#x3053;&#x3055;&#x306A;&#x3044;&#x3068;&#x3001;ACV&#x3068;&#x306E;&#x6BD4;&#x8F03;&#x3067;&#x306F;&#x3001;2&#x3064;&#x306E;&#x85AC;&#x7269;&#x306B;&#x3088;&#x3063;&#x3066;&#x751F;&#x6210;&#x3055;&#x308C;&#x305F;RNA&#x306E;&#x767A;&#x73FE;&#x30D7;&#x30ED;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x306F;&#x307E;&#x3063;&#x305F;&#x304F;&#x7570;&#x306A;&#x308B;&#x3082;&#x306E;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x7D50;&#x679C;&#x306F;&#x3001;2&#x3064;&#x306E;&#x85AC;&#x5264;&#x306E;&#x4F5C;&#x7528;&#x306E;&#x7570;&#x306A;&#x308B;&#x30E2;&#x30FC;&#x30C9;&#x3092;&#x5F37;&#x8ABF;&#x3057;&#x3001;MBV&#x306E;&#x30A2;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x306F;&#x8907;&#x6570;&#x306E;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x86CB;&#x767D;&#x8CEA;&#x3068;BGLF4&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3092;&#x4ECB;&#x3057;&#x3066;EBV&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x8EE2;&#x5199;&#x306B;&#x9593;&#x63A5;&#x7684;&#x306A;&#x5F71;&#x97FF;&#x306B;&#x30EA;&#x30F3;&#x30AF;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x305F;&#x3002;

Maribavir&#x306F;&#x3001;&#x30A6;&#x30A4;&#x30EB;&#x30B9;DNA&#x8907;&#x88FD;&#x306B;&#x52A0;&#x3048;&#x3066;&#x3001;Epstein-Barr&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x8EE2;&#x5199;&#x3092;&#x963B;&#x5BB3;&#x3059;&#x308B;

&#xB9CE;&#xC740; &#xC57D;&#xBB3C;&#xC740; &#xC140; &#xBB38;&#xD654; &#xC2DC;&#xC2A4;&#xD15C;&#xC5D0; &#xC5E1; &#xC2A4;&#xD0C0;&#xC778;-&#xBC14; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; (EBV)&#xC758; &#xBCF5;&#xC81C;&#xB97C; &#xC5B5;&#xC81C;, &#xBE44;&#xB85D; &#xC5EC;&#xC804;&#xD788; &#xD6A8;&#xACFC;&#xC801; &#xC774;&#xBA70; &#xAE30;&#xBCF8; EBV &#xAC10;&#xC5FC;&#xC5D0; &#xC0AC;&#xC6A9; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC2B9;&#xC778; &#xB41C; &#xC544;&#xBB34; &#xC57D;&#xBB3C;&#xC774; &#xC774;&#xB2E4;. &#xB354; &#xCD5C;&#xADFC;&#xC5D0;, maribavir (MBV) l ribofuranoside benzimidazole EBV &#xBCF5;&#xC81C;&#xC758; &#xAC15;&#xB825; &#xD558; &#xACE0; &#xBB34; &#xB3C5;&#xC131; &#xC5B5;&#xC81C; &#xBB3C; &#xADF8;&#xB9AC;&#xACE0; &#xD589;&#xB3D9; &#xAF64; EBV&#xC640; &#xC778;&#xAC04;&#xC758; &#xC138;&#xD3EC; &#xB2E8;&#xBC31;&#xC9C8; kinases &#xC5EC; &#xB300;&#xC0C1; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xC778; &#xC0B0;&#xD654;&#xB97C; &#xCC28;&#xB2E8; &#xD558;&#xB294; MBV&#xC758; &#xB2A5;&#xB825;&#xC5D0; &#xC8FC;&#xB85C; &#xAE30;&#xCD08;&#xD55C; acyclovir (ACV) &#xAC19;&#xC740; &#xBE44; &#xD658; &#xC2DD; &#xB274; &#xD074;&#xB808; &#xC624; &#xC2DC;&#xB4DC; &#xC720;&#xC0AC; &#xCCB4;&#xC758; &#xACE0;&#xC720; &#xBAA8;&#xB4DC;&#xB97C;&#xAC00;&#xC9C0;&#xACE0; &#xD45C;&#xC2DC; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xC57D;&#xBB3C;&#xC758; &#xD56D; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC774; &#xBCF5;&#xC7A1; &#xD55C; &#xB54C;&#xBB38;&#xC5D0;, &#xC6B0;&#xB9AC; &#xC591;&#xC801; &#xC2E4;&#xC2DC;&#xAC04; &#xB9AC;&#xBC84;&#xC2A4; &#xC804;&#xC0AC; PCR &#xAE30;&#xBC18; &#xBC30;&#xC5F4; &#xBAA8;&#xB4E0; EBV &#xC720;&#xC804;&#xC790;&#xC758; RNA &#xC2DD; &#xC218;&#xC900;&#xC5D0; MBV&#xC758; &#xD6A8;&#xACFC;&#xC758; &#xD3EC;&#xAD04;&#xC801;&#xC778; &#xBD84;&#xC11D;&#xC744; &#xC218;&#xD589; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. ACV, &#xBE44;&#xAD50;&#xC5D0;&#xC11C; &#xBCF4;&#xC5EC;&#xC8FC;&#xB294; &#xC6B0;&#xB9AC; &#xB450; &#xC57D;&#xBB3C;&#xC5D0; &#xC758;&#xD574; &#xC0DD;&#xC0B0; RNA &#xC2DD; &#xD504;&#xB85C; &#xD30C;&#xC77C;&#xC740; &#xC5EC;&#xB7EC; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC131; Mrnas&#xC758; &#xBC1C;&#xC74C;&#xB41C; &#xC800;&#xD574;&#xB97C; &#xC77C;&#xC73C;&#xD0A4;&#xB294; MBV&#xC640; ACV &#xAC70;&#xC758; &#xC5C6;&#xC74C;&#xC758; &#xC6D0;&#xC778;&#xC774; &#xC644;&#xC804;&#xD788; &#xB2E4;&#xB978;. &#xACB0;&#xACFC; &#xB450; &#xC57D;&#xBB3C;&#xC758; &#xD589;&#xB3D9;&#xC758; &#xB2E4;&#xC591; &#xD55C; &#xBAA8;&#xB4DC;&#xB97C; &#xAC15;&#xC870; &#xD558; &#xACE0; MBV &#xC791;&#xC5C5; &#xC5EC;&#xB7EC; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC131; &#xB2E8;&#xBC31;&#xC9C8; bglf4&#xC758; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC744; &#xD1B5;&#xD574; EBV &#xC720;&#xC804;&#xC790;&#xC758; &#xC804;&#xC0AC;&#xC5D0; &#xB300; &#xD55C; &#xAC04;&#xC811; &#xD6A8;&#xACFC;&#xC5D0; &#xC5F0;&#xACB0; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;.

Maribavir &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC131; DNA &#xBCF5;&#xC81C; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; &#xC5E1; &#xC2A4;&#xD0C0;&#xC778;-&#xBC14; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC804;&#xC0AC;&#xB97C; &#xC5B5;&#xC81C; &#xD55C;&#xB2E4;.

Aunque muchos f&#xE1;rmacos inhiben la replicaci&#xF3;n del virus Epstein-Barr (EBV) en sistemas de cultivo celular, a&#xFA;n existe ning&#xFA;n f&#xE1;rmaco que sea eficaz y aprobado para uso en la infecci&#xF3;n primaria por EBV. M&#xE1;s recientemente, maribavir (APV), un bencimidazol L-ribofuranosido, se ha demostrado que es un inhibidor potente y no t&#xF3;xica de la replicaci&#xF3;n de EBV y tener un modo de acci&#xF3;n muy distinta de la de los an&#xE1;logos de nucle&#xF3;sidos ac&#xED;clicos tales como el aciclovir (ACV) que es basa principalmente en la capacidad de APV para bloquear la fosforilaci&#xF3;n de las prote&#xED;nas diana por EBV y humanos prote&#xED;nas quinasas citomegalovirus. Sin embargo, ya que los mecanismos antivirales de la droga son complejos, hemos llevado a cabo un an&#xE1;lisis exhaustivo de los efectos de la APV en los niveles de expresi&#xF3;n de ARN de todos los genes del VEB con una cuantitativa en tiempo real de PCR con transcripci&#xF3;n inversa basada en la matriz. Se demuestra que en las comparaciones con ACV, los perfiles de expresi&#xF3;n de ARN producidos por los dos f&#xE1;rmacos son totalmente diferentes, con MBV causando una marcada inhibici&#xF3;n de m&#xFA;ltiples ARNm virales y con ACV causan pr&#xE1;cticamente ninguna. Los resultados ponen de manifiesto los diferentes modos de acci&#xF3;n de los dos f&#xE1;rmacos, y sugieren que la acci&#xF3;n de la APV puede estar relacionado con efectos indirectos en la transcripci&#xF3;n de los genes del EBV a trav&#xE9;s de la interacci&#xF3;n de BGLF4 con m&#xFA;ltiples prote&#xED;nas virales.

Maribavir Inhibe virus de Epstein-Barr Virus Transcripci&#xF3;n Adem&#xE1;s de la replicaci&#xF3;n del ADN viral

Maribavir (MBV) inhibits Epstein-Barr virus (EBV) replication and the enzymatic activity of the viral protein kinase BGLF4. MBV also inhibits expression of multiple EBV transcripts during EBV lytic infection. Here we demonstrate, with the use of a BGLF4 knockout virus, that effects of MBV on transcription take place primarily through inhibition of BGLF4. MBV inhibits viral genome copy numbers and infectivity to levels similar to and exceeding levels produced by BGLF4 knockout virus.

Fu-Zhang Wang

David H. Murdock Research Institute

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood