Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Feb;82(4):1884-98.
doi: 10.1128/JVI.02550-06. Epub 2007 Dec 5.

Functional analysis of the murine cytomegalovirus chemokine receptor homologue M33: ablation of constitutive signaling is associated with an attenuated phenotype in vivo

Ruth Case  1 , Emma Sharp, Tau Benned-Jensen, Mette M Rosenkilde, Nicholas Davis-Poynter, Helen E Farrell
Affiliations

Functional analysis of the murine cytomegalovirus chemokine receptor homologue M33: ablation of constitutive signaling is associated with an attenuated phenotype in vivo

Ruth Case et al. J Virol. 2008 Feb.

Abstract

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.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Genetic organization of the M33 region of the MCMV genome (BglII fragment) showing the location of restriction enzyme sites used for cloning in this study (not to scale). The HindIII site was introduced by site-directed mutagenesis.
FIG. 2.
FIG. 2.
Serpentine model of MCMV M33 indicating positions of mutations. The predicted topology of M33 within the cell membrane is shown, with shading indicating TMs I to VII and the eighth predicted α-helix. The M33 sequence shown is of the K181 (Perth) strain used in this study (GenPept accession number CAP08079). This sequence differs by two conservative amino acid substitutions relative to the prototypic Smith strain (GenPept accession number Q83207), namely L108 → M and M319 → L. N- and C-terminal truncation mutants characterized in this study are indicated with gray bars; point mutations are circled in black. The location of the 7TMR-conserved DRY motif (NRY in M33) is indicated by the box. Positions of residues conserved among rhodopsin-like 7TMR are indicated as white letters within gray circles.
FIG. 3.
FIG. 3.
Expression characteristics of wt M33, wt UL33, and M33 mutants. HEK-293 cells were transiently transfected with plasmids expressing GFP-tagged wt M33 or wt UL33 (A), the C- or N-terminal truncation M33 mutants (B), the TM II M33 mutants (C), or the TM III M33 mutants (D). Fluorescence was visualized by confocal microscopy at 24 h posttransfection with the cell surface shown by the reactivity of Alexa Fluor 594-conjugated wheat germ agglutinin (red, middle panels), the distribution of the M33 constructs shown by EGFP fluorescence (green, left panels), and the colocalization of the expressed M33 constructs with the cell surface (yellow, right panels). Scale bar, 10 μm.
FIG. 4.
FIG. 4.
CREB-mediated transcription (A, C, and E) and NFAT-mediated transcription (B, D, and F) stimulated by wt M33 and mutant derivatives. HEK-293 cells were transiently transfected with either the C- or N-terminal M33 truncation mutants (A and B), the TM II M33 mutants (C and D), or the TM III M33 mutants (E and F) at the various doses shown, together with luciferase reporter constructs. Results are expressed as relative light units (RLU) compared to the negative control plasmid pcDNA3. wt M33 was included as a reference in each assay. Mutants which were negative for cell surface distribution are shown as gray symbols with dotted lines, and mutants which were positive for cell surface distribution are shown as black open or crossed symbols with solid lines; the symbols are identified on the figure. The mean and standard error are shown (n = 8 to 16).
FIG. 5.
FIG. 5.
Activation of PLC by cell surface-expressed receptors. HEK-293 cells were transiently transfected with selected M33 mutants (cell surface expressed), alongside wt M33 and pcDNA3. Measurements of [myo-3H]inositol phosphorylation stimulated by the various constructs are expressed as relative cpm (Rcpm) compared to pcDNA3. The mean and standard error are shown (n = 9).
FIG. 6.
FIG. 6.
Western blotting and total protein expression analysis of GFP-tagged wt M33 and mutated M33 derivatives. (A to D) Western blotting samples were prepared from COS-7 cell lysates 24 h posttransfection and immunoblotted using an anti-EGFP monoclonal antibody. Blots were derived from separate experiments, with wt M33-transfected cells included on each occasion for comparison purposes. (E and F) Total protein expression. HEK-293 cells were transfected with the various GFP-tagged mutants, alongside wt M33 and pcDNA3. Mutants which were negative for cell surface distribution are shown as gray symbols with dotted lines, and mutants which were positive for cell surface distribution are shown as black open or crossed symbols with solid lines; the symbols are specified in the figure. GFP fluorescence was determined and is expressed as relative light units (RLU) compared with the negative control (pcDNA3). The mean and standard error are shown (n = 4).
FIG. 7.
FIG. 7.
Multistep in vitro growth curve of wt MCMV and M33 mutants. Subconfluent cultures of NIH-3T3 cells were prepared in six-well dishes and infected with each of the MCMV recombinants or wt MCMV at an MOI of 0.01. Infected culture medium (cell free) was harvested daily for 5 days and stored at −80°C until quantification by plaque assay on MEFs. The mean and standard errors are shown (duplicate cultures were titrated for each virus). Panels A and B comprise two separate experiments, characterizing different sets of viruses. In panel A, the symbols are as follows: filled triangles, wt MCMV; open circles, M33Δi; open triangles, M33ΔC24; crosses, M33ΔC38; filled diamonds, M33(F79D); stars, M33(N130D); and open squares, UL33. In panel B the wt MCMV is represented by filled triangles, and two independent M33(R131Q) transfectants are represented by open diamonds and open squares. p.i., postinfection.
FIG. 8.
FIG. 8.
Expression of GFP-tagged wt M33 and mutated derivatives by recombinant MCMV. Subconfluent monolayers of MEFs were prepared in 96-well trays and infected at an MOI of 3 with MCMV recombinants expressing GFP-tagged wt M33 or selected mutants (F79D, R131Q, or ΔC38). K181 (Perth) was included as a GFP-negative control. At 15 h postinfection, M33-GFP expression was quantitated by immunofluorescence using a rabbit polyclonal anti-GFP antiserum, expressed as relative light units (RLU) compared with the negative control (K181). The mean and standard error are shown (n = 6).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Baldwin, J. M. 1993. The probable arrangement of the helices in G protein-coupled receptors. EMBO J. 121693-1703. - PMC - PubMed
    1. Ballesteros, J., S. Kitanovic, F. Guarnieri, P. Davies, B. J. Fromme, K. Konvicka, L. Chi, R. P. Millar, J. S. Davidson, H. Weinstein, and S. C. Sealfon. 1998. Functional microdomains in G protein-coupled receptors. The conserved arginine-cage motif in the gonadotropin-releasing hormone receptor. J. Biol. Chem. 27310445-10453. - PubMed
    1. Ballesteros, J. A., A. D. Jensen, G. Liapakis, S. G. Rasmussen, L. Shi, U. Gether, and J. A. Javitch. 2001. Activation of the beta 2-adrenergic receptor involves disruption of an ionic lock between the cytoplasmic ends of transmembrane segments 3 and 6. J. Biol. Chem. 27629171-29177. - PubMed
    1. Beisser, P. S., C. Vink, J. G. Van Dam, G. Grauls, S. J. Vanherle, and C. A. Bruggeman. 1998. The R33 G protein-coupled receptor gene of rat cytomegalovirus plays an essential role in the pathogenesis of viral infection. J. Virol. 722352-2363. - PMC - PubMed
    1. Bentz, G. L., M. Jarquin-Pardo, G. Chan, M. S. Smith, C. Sinzger, and A. D. Yurochko. 2006. Human cytomegalovirus (HCMV) infection of endothelial cells promotes naive monocyte extravasation and transfer of productive virus to enhance hematogenous dissemination of HCMV. J. Virol. 8011539-11555. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources