Use of a Murine Cytomegalovirus K181-Derived Bacterial Artificial Chromosome as a Vaccine Vector for Immunocontraception

Redwood, Alec; Messerle, Martin; Harvey, Naomi D.; Hardy, Christopher M.; Koszinowski, Ulrich H.; Lawson, Malcolm; Shellam, Geoffrey

doi:10.1128/jvi.79.5.2998-3008.2005

Cited by 75 publications

(72 citation statements)

References 55 publications

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“…We used recombinant DNA techniques to generate a MCMV (K181 strain) mutant with the entire M33 open reading frame (ORF) (⌬M33) deleted or MCMV mutants expressing either the M33FLAG (WT) or M33FLAG(R131A) mutant (see Fig. S1 in the supplemental material) (61). Using this strategy, the M33FLAG (WT) and M33FLAG(R131A) viruses represent "rescues" of the ⌬M33 virus.…”

Section: Resultsmentioning

confidence: 99%

Activation of Intracellular Signaling Pathways by the Murine Cytomegalovirus G Protein-Coupled Receptor M33 Occurs via PLC-β/PKC-Dependent and -Independent Mechanisms

Sherrill

Stropes

Schneider

et al. 2009

J Virol

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The presence of numerous G protein-coupled receptor (GPCR) homologs within the herpesvirus genomes suggests an essential role for these genes in viral replication in the infected host. Such is the case for murine cytomegalovirus (MCMV), where deletion of the M33 GPCR or replacement of M33 with a signaling defective mutant has been shown to severely attenuate replication in vivo. In the present study we utilized a genetically altered version of M33 (termed R131A) in combination with pharmacological inhibitors to further characterize the mechanisms by which M33 activates downstream signaling pathways. This R131A mutant of M33 fails to support salivary gland replication in vivo and, as such, is an important tool that can be used to examine the signaling activities of M33. We show that M33 stimulates the transcription factor CREB via heterotrimeric G q/11 proteins and not through promiscuous coupling of M33 to the G s pathway. Using inhibitors of signaling molecules downstream of G q/11 , we demonstrate that M33 stimulates CREB transcriptional activity in a phospholipase C-␤ and protein kinase C (PKC)-dependent manner. Finally, utilizing wild-type and R131A versions of M33, we show that M33-mediated activation of other signaling nodes, including the mitogenactivated protein kinase family member p38␣ and transcription factor NF-B, occurs in the absence of G q/11 and PKC signaling. The results from the present study indicate that M33 utilizes multiple mechanisms to modulate intracellular signaling cascades and suggest that signaling through PLC-␤ and PKC plays a central role in MCMV pathogenesis in vivo.

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Section: Resultsmentioning

confidence: 99%

Activation of Intracellular Signaling Pathways by the Murine Cytomegalovirus G Protein-Coupled Receptor M33 Occurs via PLC-β/PKC-Dependent and -Independent Mechanisms

Sherrill

Stropes

Schneider

et al. 2009

J Virol

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“…Bacterial artificial chromosome derived wild-type MCMV (strain K181-Perth) and MHV68 WUMS [obtained from American Type Culture Collection (ATCC) #VR1465] were generated on NIH 3T3 fibroblasts (ATCC, #CRL-1658) as previously described (39,40). Viral stocks were propagated, clarified, and concentrated as previously described (41).…”

Section: Methodsmentioning

confidence: 99%

Electrochemical detection of a single cytomegalovirus at an ultramicroelectrode and its antibody anchoring

Dick

Hilterbrand

Boika

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

147

132

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We report observations of stochastic collisions of murine cytomegalovirus (MCMV) on ultramicroelectrodes (UMEs), extending the observation of discrete collision events on UMEs to biologically relevant analytes. Adsorption of an antibody specific for a virion surface glycoprotein allowed differentiation of MCMV from MCMV bound by antibody from the collision frequency decrease and current magnitudes in the electrochemical collision experiments, which shows the efficacy of the method to size viral samples. To add selectivity to the technique, interactions between MCMV, a glycoprotein-specific primary antibody to MCMV, and polystyrene bead "anchors," which were functionalized with a secondary antibody specific to the Fc region of the primary antibody, were used to affect virus mobility. Bead aggregation was observed, and the extent of aggregation was measured using the electrochemical collision technique. Scanning electron microscopy and optical microscopy further supported aggregate shape and extent of aggregation with and without MCMV. This work extends the field of collisions to biologically relevant antigens and provides a novel foundation upon which qualitative sensor technology might be built for selective detection of viruses and other biologically relevant analytes.collisions | cytomegalovirus | electrochemistry | murine cytomegalovirus | virus O ver the past decade, the study of discrete collision events on ultramicroelectrodes (UMEs) has gained attention due to the interest in understanding stochastic phenomena by electrochemistry. By observing the collisions of small particles, there is the possibility that information can be deduced that is not available in ensemble measurements. The electrochemical study of single collision events has been applied to a wide range of hard nanoparticles (NPs), which include metal, metal oxide, and organic NPs [platinum (1), silver (2), gold (3), nickel (4), copper (5), iridium oxide (6), cerium oxide (7), titanium oxide (8), silicon oxide (9), indigo (10), polystyrene (11), and relatively large aggregates of fullerene (12)]. Recently, collisions of soft particles have been investigated, such as toluene droplets (13) and liposomes (14). Also, collisions of toluene and tri-n-propylamine droplets were observed simultaneously by both electrochemical and electrogenerated chemiluminescent (ECL) measurements (15).Similarly, a variety of techniques have been developed to observe these collision events. The interested reader can consult the references for a discussion on each of the techniques used to observe stochastic events electrochemically: blocking (9, 13), electrocatalytic amplification (1), open circuit potential (16), droplet blocking/reactor (13,14), and ECL (15,17,18). The simplest and most reproducible method of observing collisions is a technique termed blocking, which is so named because particles, which are brought to the electrode by a diffusion-limited flux and/or electrophoretic migration, irreversibly adsorb (1) to the electrode surface, blocking the flux of red...

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“…The availability of whole viral genomes packaged within bacterial artificial chromosomes (BACs) and the ability to generate targeted mutations through DNA recombination are becoming useful tools to study viral GPCRs in this context. For example, the recent development of a BAC containing the full genome of the K181 strain of MCMV will allow for manipulation of the M33 ORF to dissect the signaling activities (G protein-dependent or G protein-independent) and the desensitization properties of M33 that might be involved in viral pathogenesis in vivo (Redwood et al, 2005). Given the functional similarities in signaling activity and regulation by GRK and arrestin proteins as discussed above, it is likely that M33 and US28 share related roles in the pathogenesis of their respective viruses.…”

Section: Murine Herpesviruses As Models For In Vivo Infectionmentioning

confidence: 99%

Desensitization of herpesvirus-encoded G protein-coupled receptors

Sherrill¹,

Miller²

2008

Life Sciences

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Members of the herpesvirus family, including human cytomegalovirus (HCMV) and Kaposi's Sarcoma-associated herpesvirus (KSHV/HHV-8), encode G protein-coupled receptor (GPCR) homologs, which strongly activate classical G-protein signal transduction networks within the cell. In animal models of herpesvirus infection, the viral GPCRs appear to play physiologically important roles by enabling viral replication within tropic tissues and by promoting reactivation from latency. While a number of studies have defined intracellular signaling pathways activated by herpesviral GPCRs, it remains unclear if their physiological function is subjected to the process of desensitization as observed for cellular GPCRs. G protein-coupled receptor kinases (GRK) and arrestin proteins have been recently implicated in regulating viral GPCR signaling; however, the role that these desensitization proteins play in viral GPCR function in vivo remains unknown. Here, we review what is currently known regarding viral GPCR desensitization and discuss potential biological ramifications of viral GPCR regulation by the host cell desensitization machinery.

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Use of a Murine Cytomegalovirus K181-Derived Bacterial Artificial Chromosome as a Vaccine Vector for Immunocontraception

Cited by 75 publications

References 55 publications

Activation of Intracellular Signaling Pathways by the Murine Cytomegalovirus G Protein-Coupled Receptor M33 Occurs via PLC-β/PKC-Dependent and -Independent Mechanisms

Activation of Intracellular Signaling Pathways by the Murine Cytomegalovirus G Protein-Coupled Receptor M33 Occurs via PLC-β/PKC-Dependent and -Independent Mechanisms

Electrochemical detection of a single cytomegalovirus at an ultramicroelectrode and its antibody anchoring

Desensitization of herpesvirus-encoded G protein-coupled receptors

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