Systematic mutagenesis of the murine gammaherpesvirus 68 M2 protein identifies domains important for chronic infection - PubMed (original) (raw)

Systematic mutagenesis of the murine gammaherpesvirus 68 M2 protein identifies domains important for chronic infection

Jeremy H Herskowitz et al. J Virol. 2008 Apr.

Abstract

Murine gammaherpesvirus 68 (MHV68) infection of inbred mice represents a genetically tractable small-animal model for assessing the requirements for the establishment of latency, as well as reactivation from latency, within the lymphoid compartment. By day 16 postinfection, MHV68 latency in the spleen is found in B cells, dendritic cells, and macrophages. However, as with Epstein-Barr virus, by 3 months postinfection MHV68 latency is predominantly found in isotype-switched memory B cells. The MHV68 M2 gene product is a latency-associated antigen with no discernible homology to any known cellular or viral proteins. However, depending on experimental conditions, the M2 protein has been shown to play a critical role in both the efficient establishment of latency in splenic B cells and reactivation from latently infected splenic B cells. Inspection of the sequence of the M2 protein reveals several hallmarks of a signaling molecule, including multiple PXXP motifs and two potential tyrosine phosphorylation sites. Here, we report the generation of a panel of recombinant MHV68 viruses harboring mutations in the M2 gene that disrupt putative functional motifs. Subsequent analyses of the panel of M2 mutant viruses revealed a functionally important cluster of PXXP motifs in the C-terminal region of M2, which have previously been implicated in binding Vav proteins (P. A. Madureira, P. Matos, I. Soeiro, L. K. Dixon, J. P. Simas, and E. W. Lam, J. Biol. Chem. 280:37310-37318, 2005; L. Rodrigues, M. Pires de Miranda, M. J. Caloca, X. R. Bustelo, and J. P. Simas, J. Virol. 80:6123-6135, 2006). Further characterization of two adjacent PXXP motifs in the C terminus of the M2 protein revealed differences in the functions of these domains in M2-driven expansion of primary murine B cells in culture. Finally, we show that tyrosine residues 120 and 129 play a critical role in both the establishment of splenic latency and reactivation from latency upon explant of splenocytes into tissue culture. Taken together, these analyses will aide future studies for identifying M2 interacting partners and B-cell signaling pathways that are manipulated by the M2 protein.

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Figures

FIG. 1.

FIG. 1.

Deduced amino acid sequence of the M2 protein, with PXXP motifs and candidate tyrosine phosphorylation sites indicated in bold. Each PXXP motif is numbered, and the mutations introduced into each motif are indicated above the M2 sequence. The mutations of proline residues 70, 71, and 73 disrupt both the P3 and P4 PXXP motifs. Also shown are the mutations introduced into the tyrosine residues at positions 120 and 129, which were mutated to either phenylalanine or aspartic acid. The P7 PXXP motif is the only consensus class I SH3 binding motif (K/RxxPxxP) present in the M2 protein.

FIG. 2.

FIG. 2.

Construction of M2 mutant viruses and corresponding marker rescue viruses. (A) The MHV68 genome cloned as a BAC (2) was used to generate all M2 recombinant viruses by RecA-mediated recombination. For M2 mutant viruses exhibiting robust establishment-of-latency and/or reactivation-from-latency phenotypes, a marker rescue virus in which the wt M2 sequences were targeted to the M2 ORF by allelic exchange to restore the wt phenotype was generated. The DNA probe used for the Southern blot analyses contained sequence from the M2 ORF (genomic coordinates, bp 4031 to 4627). Relevant restriction sites are indicated. A, AluI; Bg, BglI; D, DdeI; N, NcoI; BU, BstUI; Bs, BssHII. (B and C) Southern blot analyses of recombinant viruses harboring mutations in the indicated PXXP motifs of M2, along with corresponding marker rescue viruses. (D) Southern blot analyses of recombinant viruses harboring mutations in the indicated tyrosine residues of M2 and their corresponding marker rescue viruses. (E) Southern blot analysis of M2.Stop(2). 32P-labeled molecular size standards (Lambda DNA-BstEII digest; New England Biolabs, Beverly, MA) were included in each Southern blot analysis. WT, wt MHV68; MR, marker rescue virus.

FIG. 3.

FIG. 3.

Expression of mutant M2 proteins in mammalian cells. (A) Schematic illustration of the locations of PXXP motifs and tyrosine residues in the M2 protein which have been targeted for mutation in this report. (B) Cos-1 cells were transiently transfected with plasmids expressing the indicated forms of mutant M2 protein. At 48 hours posttransfection, cells were harvested and M2 immunoprecipitations were performed as described in Materials and Methods. (C) Cos-1 cells were transiently transfected with plasmids expressing the indicated forms of mutant M2 protein and GFP. At 24 hours posttransfection, 50 μg/ml CHX (+) or a vehicle control (−) was added to each sample, and cell lysates were harvested at the indicated times, in hours, posttreatment. Protein half-life was calculated by determining the time point at which the band intensity ratio of M2 to GFP was 50% of this ratio at time zero. WT, wt M2; Cntl, vector control; α, anti; IP, immunoprecipitation; _t_1/2, half-life.

FIG. 4.

FIG. 4.

Identification of PXXP motifs and tyrosine residues in the M2 protein that are involved in establishment of and/or reactivation from MHV68 latency in the spleen. C57BL/6J mice were infected intranasally with 100 PFU of wt MHV68, a recombinant M2 mutant virus, or its corresponding marker rescue virus, and spleens were harvested at 16 dpi. Bulk splenocytes were analyzed by limiting-dilution ex vivo reactivation assays and limiting-dilution viral genome PCR assays, as described in Materials and Methods. (A, C, E, G, I, K and M) Frequency of splenocytes harboring viral genome. A limiting-dilution PCR assay was used to determine the frequencies of splenocytes harboring the viral genome. Samples were serially diluted into a background of 104 uninfected cells, lysed, and subjected to nested PCR to detect the viral genome (see Materials and Methods). (B, D, F, H, J, L, and N) Frequency of splenocytes reactivating virus. Results are shown for a limiting-dilution ex vivo reactivation assay in which intact splenocytes were serially diluted on MEF indicator monolayers in parallel with mechanically disrupted samples (to distinguish between virus reactivating from latency and preformed infectious virus). In this report, little or no preformed infectious virus was detected for any of the viruses analyzed (data not shown). For each sample dilution, 24 wells were scored for the presence of CPE (see Materials and Methods). Data are expressed as the mean percentages of wells positive for virus (CPE or viral DNA) ± the standard errors of the means. Curve fit lines for both assays were derived through nonlinear regression analyses. The dashed line indicates 63.2%, from which the frequency of cells reactivating virus or the frequency of cells harboring the viral genome was determined based on Poisson distribution. The data shown represent the means for at least two independent experiments. The recombinant virus nomenclature is described in the legends to Fig. 1 and 3 and Materials and Methods.

FIG. 5.

FIG. 5.

M2 recombinant viruses display normal acute-phase replication in vivo following intranasal inoculation. C57BL/6J mice were infected intranasally with 100 PFU of a recombinant M2 mutant virus or its corresponding marker rescue virus, and the left lung was extracted at 9 dpi (d9). (A) M2 recombinant viruses with site-directed mutations in PXXP motifs and the corresponding marker rescue viruses. (B) M2 recombinant viruses with site-directed mutations in tyrosine residues and the corresponding marker rescue viruses. The results shown were compiled from two independent experiments with three mice per experiment (each symbol represents data from an individual mouse). Virus titers in lungs were determined by a plaque assay on NIH 3T12 monolayers as described in Materials and Methods. The dashed line indicates the level of detection of the plaque assay (50 PFU), and the solid line indicates the mean virus titer of each group. The recombinant virus nomenclature is described in the legends to Fig. 1 and 3 and Materials and Methods.

FIG. 6.

FIG. 6.

M2 expression in primary B cells results in expansion of transduced population. (A) Representative flow cytometry data from 3 days posttransduction. (B) M2-expressing B cells expand in culture over time. Triplicate B-cell cultures were transduced with wt M2 or the indicated M2 mutant retrovirus and allowed to rest for 48 h before analysis. Cells were stained with anti-Thy1.1 and analyzed daily for Thy1.1-positive cells. Trypan blue exclusion was used to count absolute numbers of live and dead cells in triplicate wells every day; notably, absolute numbers of cells per well were unchanged over time. Data are representative of two independent experiments.

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