Constitutive Signaling of the Human Cytomegalovirus-encoded Receptor UL33 Differs from That of Its Rat Cytomegalovirus Homolog R33 by Promiscuous Activation of G Proteins of the Gq, Gi, and Gs Classes

Casarosa, Paola; Gruijthuijsen, Yvonne K.; Michel, Detlef; Beisser, Patrick S.; Holl, Jens; Fitzsimons, Carlos P.; Verzijl, Dennis; Bruggeman, Cathrien A.; Mertens, Thomas; Leurs, Rob; Vink, Cornelis; Smit, Martine J.

doi:10.1074/jbc.m306530200

Cited by 84 publications

(73 citation statements)

References 50 publications

(75 reference statements)

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“…In summary, similar to previous studies, the data here demonstrate that the HCMV vGPCR US28 and UL33 have the propensity to alter cellular migration (Hjorto et al, 2013;Vomaske et al, 2009) and interfere with transcription (Boomker et al, 2006;Casarosa et al, 2003;Maussang et al, 2009;McLean et al, 2004;Waldhoer et al, 2002;Wen et al, 2009) in several cell types. In this study, we…”

Section: Discussionsupporting

confidence: 89%

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Cell-type specific activities of cytomegalovirus GPCR homologues

Oliveira¹

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The human cytomegalovirus (HCMV) is the main agent of congenital infection worldwide. Although primary infections or reactivations do not represent a threat to most individuals, they impose a lifethreating event to a developing foetus or immunocompromised individuals. Although we have learnt much about sites of viral replication and cells involved in the end-stage disease, the specific pathway and essential cell types that the virus must go through to later establish a successful infection and latency is still not clear. HCMV encodes four GPCR homologues (vGPCR): US27, US28, UL33 and UL78. vGPCR have been implicated in viral pathogenesis due to their ability to activate signalling pathways and, thus, alter physiological processes to favour infection. US28 and UL33 have been shown to activate several kinases and transcriptional factors. A key component of both US28 and UL33 sequence is the DRY (Asp/Arg/Tyr) motif, a region directly involved with G protein binding.Because Gα protein content is cell type-dependent, signalling activation by vGPCR can differ from cell to cell. Furthermore, these receptors share the particular characteristic of being constitutively Functional studies demonstrated migration induction of HTR-8 following transfection by either US28 or UL33. In order to investigate potential mechanisms, signalling pathways were probed via detection of activated kinases and the secretion of matrix metalloproteinases (MMP) and chemokines (CK) via luminex assays. Although differences in the level of kinases could not be measured by western blot, induction of MMP and CK were detected by Luminex assays, with contrasting results for US28 and UL33 in transfected cells (HTR-8 and HUVEC). An HCMV recombinant disrupted for US28 signalling (DQY) was generated and compared with wild type HCMV and a US28 null mutant for induction of MMP and CK in virus-infected cells. US28-dependent effects were identified for infected human foreskin fibroblasts, but results for HUVEC were equivocal.iii To determine potential bottlenecks to viral dissemination that may require M33 function(s), mice were infected with M33 knockout (M33) viruses by different routes: intranasal, -mimicking the most natural route, footpad -a more compartmentalized route, and intraperitoneal, the most direct route to major organs. To help track viral dissemination, a luciferase tagged M33 was generated and infection was compared to control virus (M78luc). M33 intranasal infection did not result in attenuation for colonisation of the nose, draining (superficial cervical) lymph nodes (dLN) or the lungs. Via footpad route, M33 colonised the footpad and dLN to levels equivalent to control virus, but was significantly attenuated in spread to the spleen and, subsequently, the salivary glands.Following intraperitoneal infection, the virus is readily delivered to major organs due to direct access to the bloodstream, there was no difference in the colonisation of primary organs (spleen, liver) between M33 and control MCMV, but M33 was still unable ...

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Section: Discussionsupporting

confidence: 89%

“…US28 and UL33 have been described to subvert several physiological pathways (Casarosa et al, 2001;Waldhoer et al 2002;Casarosa et al, 2003). Alteration of cellular properties, such as migration, represent a tangible outcome as how these changes may contribute to viral pathogenesis.…”

Section: Discussionmentioning

confidence: 99%

Cell-type specific activities of cytomegalovirus GPCR homologues

Oliveira¹

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“…Unlike cellular CC chemokine receptors, UL33, M33, and R33 demonstrate constitutive activity in multiple signaling pathways (10,32,68). Studies of MCMV demonstrated that although M33 is dispensable for virus replication in vitro, the absence of M33 renders MCMV unable to replicate in, or disseminate to, the salivary glands following infection of mice (16).…”

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confidence: 99%

Functional Analysis of the Murine Cytomegalovirus Chemokine Receptor Homologue M33: Ablation of Constitutive Signaling Is Associated with an Attenuated Phenotype In Vivo

Case

Sharp

Benned-Jensen

et al. 2008

J Virol

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The murine cytomegalovirus (MCMV) M33 gene is conserved among all betaherpesviruses and encodes a homologue of seven-transmembrane receptors (7TMR) with the capacity for constitutive signaling. Previous studies have demonstrated that M33 is important for MCMV dissemination to or replication within the salivary glands. In this study, we probed N-and C-terminal regions of M33 as well as known 7TMR signature motifs in transmembrane (TM) II and TM III to determine the impact on cell surface expression, constitutive signaling, and in vivo phenotype. The region between amino acids R 340 and A 353 of the C terminus was found to be important for CREB-and NFAT-mediated signaling, although not essential for phosphatidylinositol turnover. Tagging or truncation of the N terminus of M33 resulted in loss of cell surface expression. Within TM II, an F79D mutation abolished constitutive signaling, demonstrating a role, as in other cellular and viral 7TMR, of TM II in receptor activation. In TM III, the arginine (but not the asparagine) residue of the NRY motif (the counterpart of the common DRY motif in cellular 7TMR) was found to be essential for constitutive signaling. Selected mutations incorporated into recombinant MCMV showed that disruption of constitutive signaling for a viral 7TMR homologue resulted in a reduced capacity to disseminate to or replicate in the salivary glands. In addition, HCMV UL33 was found to partially compensate for the lack of M33 in vivo, suggesting conserved biological roles of the UL33 gene family.

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“…A number of other herpesvirus 7-tm proteins exhibit either constitutive or ligand-mediated signaling characteristics (3,8,11,12,21,22,53). This prompted us to wonder whether U51 might exhibit cell signaling properties that could perhaps influence the efficiency of viral replication.…”

Section: Discussionmentioning

confidence: 99%

“…Since the ligand repertoire and signaling properties of HHV-6A U51 remain largely unknown, it is also possible that U51 may interact with other chemokines or nonchemokine ligands and/or that it may have the capacity to induce signaling events in either a ligand-specific or a constitutive fashion (akin to HCMV US28 and other herpesvirus 7-tm proteins) (3,8,11,12,22,53).…”

Section: Discussionmentioning

confidence: 99%

The Human Herpesvirus 6 G Protein-Coupled Receptor Homolog U51 Positively Regulates Virus Replication and Enhances Cell-Cell Fusion In Vitro

Zhu¹,

Bradel-Tretheway²,

Sumagin³

et al. 2005

J Virol

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Human herpesvirus 6 (HHV-6) is a ubiquitous T-lymphotropic betaherpesvirus that encodes two G proteincoupled receptor homologs, U12 and U51. HHV-6A U51 has been reported to bind to CC chemokines including RANTES, but the biological function of U51 remains uncertain. In this report, we stably expressed short interfering RNAs (siRNAs) specific for U51 in human T cells and then infected these cells with HHV-6. Viral DNA replication was reduced 50-fold by the U51 siRNA, and virally induced cytopathic effects were also inhibited. In contrast, viral replication and syncytium formation were unaltered in cells that expressed a scrambled derivative of the siRNA or an irrelevant siRNA and were restored to normal when a human codon-optimized derivative of U51 was introduced into cells containing the U51 siRNA. To examine the mechanism whereby U51 might contribute to viral replication, we explored the signaling characteristics of U51. None of the chemokines and opioids tested was able to induce G protein coupling by U51, and no evidence for opioid ligand binding by U51 was obtained. The effect of U51 on cell-cell fusion was also evaluated; these studies showed that U51 enhanced cell fusion mediated by the G protein of vesicular stomatitis virus. However, a U51-specific antiserum had no virus-neutralizing activity, suggesting that U51 may not be involved in the initial interaction between the virus particle and host cell. Overall, these studies suggest that HHV-6 U51 is a positive regulator of virus replication in vitro, perhaps because it may promote membrane fusion and facilitates cell-cell spread of this highly cell-associated virus.Human herpesvirus 6 (HHV-6) was first isolated in 1986 from patients with lymphoproliferative disorders (43) and later was identified as the causative agent of roseola infantum (56) and of acute febrile illness (41, 58) in young children. Following primary infection, the virus is able to establish a highly successful state of coexistence with the host, resulting in persistent infection with occasional but generally nonsymptomatic reactivation (13,24). However, the virus can cause rare, serious complications in immunocompromised hosts or in the context of stem cell transplantation, including encephalitis, hepatitis, and bone marrow suppression (14,54,57). There are two variants of this virus, 6A and 6B, which have characteristic differences in their cell tropism and biological properties (1, 4, 16, 44) as well as approximately 10% overall sequence divergence at the genomic level (18,23,25).The U51 gene is one of the two 7-transmembrane (7-tm) receptors carried by HHV-6 (23). It has been shown to be most closely related to the UL78 gene family from human cytomegalovirus (CMV), and gene knockout experiments using the rat CMV have revealed that this gene (R78) is necessary for efficient virus replication in vivo, suggesting that R78 (and perhaps U51 as well) may play a role in virus replication and virulence (6). Direct analyses of U51 itself have revealed that HHV-6 U51 can bind certain CC chemok...

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Constitutive Signaling of the Human Cytomegalovirus-encoded Receptor UL33 Differs from That of Its Rat Cytomegalovirus Homolog R33 by Promiscuous Activation of G Proteins of the Gq, Gi, and Gs Classes

Cited by 84 publications

References 50 publications

Cell-type specific activities of cytomegalovirus GPCR homologues

Cell-type specific activities of cytomegalovirus GPCR homologues

Functional Analysis of the Murine Cytomegalovirus Chemokine Receptor Homologue M33: Ablation of Constitutive Signaling Is Associated with an Attenuated Phenotype In Vivo

The Human Herpesvirus 6 G Protein-Coupled Receptor Homolog U51 Positively Regulates Virus Replication and Enhances Cell-Cell Fusion In Vitro

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