Previous reports implicated functional and/or physical interactions among some of the M139, M140, and/or M141 gene products (9
). To assess the nature of the physical interactions, we devised five approaches. Initially, fibroblasts or macrophages infected with WT virus or mutant MCMV having M139, M140, or M141 deleted were subjected to a series of immunoprecipitation experiments to detect coprecipitating proteins during virus infection. Secondly, coimmunoprecipitation of the viral proteins was examined in cells transiently expressing epitope-tagged M139, M140, and M141, in the absence of other viral proteins. Thirdly, M139, M140, and M141 were cotranscribed and translated in vitro to assess association of the proteins in the absence of cellular proteins. Fourthly, the subcellular location where the viral proteins reside, both individually and together, was identified by confocal microscopy. Finally, sedimentation gradient analysis was used to assess the number of complexes that exist as physical entities in the context of virus infection.
Identification of proteins coprecipitating with M139, M140, or M141 gene products.
We began by identifying protein products that coprecipitated with the M139, M140, and M141 gene products within infected cells. Immunoprecipitation assays were performed using rabbit polyclonal antisera to detect potential coprecipitation of the two M139 products (p75M139 and p61M139), pM140 (56 kDa), and pM141 (52 kDa) during the course of MCMV infection in either fibroblasts or macrophages. Note that there is negligible cross-reactivity among the three antisera for each of the protein products, as demonstrated by Western blotting (9
) and immunoprecipitation of radiolabeled proteins (as seen in Fig. ).
FIG. 2. Sequential immunoprecipitations identify binding partners in the complex. Radiolabeled proteins were immunoprecipitated from lysates of NIH 3T3 cells infected with WT MCMV (1 PFU/cell) with rabbit polyclonal antisera to M139 or M140 gene products (First (more ...)
Immunoprecipitation of the proteins from infected cells revealed several interesting patterns of coprecipitation. As shown in Fig. , the anti-M139 antisera precipitated the expected M139 gene products of 75 and 61 kDa, in addition to proteins of 98, 56, and 52 kDa (9
). The identity of the 98-kDa protein is currently unknown; however, this protein coprecipitates with anti-M139 antisera only in the context of WT virus infection. The smaller two proteins were identified as products of M140 and M141, respectively (9
). These identifications were based on molecular weights and on the fact that these protein bands were not coprecipitated with anti-M139 antisera from cells infected with mutant MCMV having M140 or M141 deleted (Fig. ).
FIG. 1. Coimmunoprecipitation reveals complex formation among M139, M140, and M141 gene products. The figure shows autoradiographs of radiolabeled proteins immunoprecipitated from lysates of NIH 3T3 cells either mock infected, infected with WT MCMV, or infected (more ...)
Anti-M141 antiserum consistently precipitated the 52-kDa pM141, as shown in Fig. . In some experiments, coprecipitation of the 56-kDa pM140 with this antiserum was also evident.
Figure also shows that anti-M140 antisera precipitated the expected product of 56 kDa in addition to the 52-kDa M141 product. The identity of the 52-kDa protein as the M141 product was confirmed by genetic analyses, which demonstrated that the 52-kDa product was not coprecipitated with anti-M140 antiserum when cells were infected with MCMV having M141 deleted (Fig. ).
These genetic analyses also demonstrated that pM140 and pM141 are able to complex in the absence of M139 gene products but that M139 products do not bind pM140 or pM141 expressed as individual proteins. Anti-M140 antiserum coprecipitated pM141 from cells infected with mutant MCMV having M139 deleted (Fig. ). However, in the absence of pM140, pM141 did not coprecipitate with anti-M139 antiserum, and likewise, in the absence of M141, pM140 did not complex with the M139 proteins (Fig. ). Our previously published data indicate that, in the absence of pM140, the half-life of pM141 is reduced from 2 to 1 h, as determined by pulse-chase experiments (9
). However, those experiments also revealed that, in the absence of a chase, pM141 is clearly detected by immunoprecipitation with anti-M141 antiserum in cells infected with RVΔ140 (9
). Therefore, newly synthesized pM141 is available as a potential binding partner for M139 gene products in the absence of pM140. Collectively, the data in Fig. indicate that (i) pM140 and pM141 form a stable complex independent of the M139 proteins and (ii) M139 proteins stably interact with pM140 and pM141 only when these binding partners are in complex.
To confirm the identity of complexes formed in the context of MCMV infection, proteins immunoprecipitated from infected cells were denatured and subsequently reimmunoprecipitated with each specific antibody. From these experiments, it was evident that anti-M139 precipitates a complex composed of M139, M140, and M141 products, while anti-M140 precipitates a complex of pM140 and pM141 that does not contain M139 proteins (Fig. ). This consistent pattern, with anti-M139 serum coprecipitating pM140 and pM141 and anti-M140 serum coprecipitating pM141, was independent of virus preparation or source of specific antisera and was evident in infected macrophages as well (data not shown). In addition, coprecipitation was not evident when proteins were denatured prior to immunoprecipitation (data not shown). Note that the relative intensities of each of the bands likely reflect immunoprecipitation of both complexed and free forms of the proteins. These data confirmed that at least two stable complexes coexist within MCMV-infected cells: a pM140/pM141 complex and one composed of products from all three genes.
Although coprecipitation of pM141 by anti-M140 serum was highly consistent, the ability to detect coprecipitation of pM140 with anti-M141 serum was variable. Thus, we considered the possibility that anti-M141 preferentially recognizes a free form of pM141, compared to the complexed form, or that the majority of pM141 exists independently of the complex. To address these possibilities, we performed a two-step immunoprecipitation assay in which infected-cell lysates were first subjected to immunoprecipitation with anti-pM141, and the supernatants from the pelleted immune complexes were then subjected to a second round of immunoprecipitations with anti-M140 or -M141 sera. The data in Fig. demonstrated coprecipitation of pM140 (56 kDa) by anti-M141 antisera from nondenatured supernatants partially depleted of pM141. Furthermore, a significant amount of pM141 was detectable in the second round of immunoprecipitations when complexed proteins remaining after the initial anti-M141 immunoprecipitation were first denatured. These data suggested that the antibody preferentially recognizes a free form of pM141. In addition, these data demonstrated that both anti-M140 and anti-M141 sera are capable of coprecipitating both pM140 and pM141 as a complex. Collectively, these data further supported the contention that pM140 and pM141 exist as a stable complex, independently from the larger pM139-associated complex, during MCMV infection.
FIG. 3. The M141 protein exists in a free and complexed form. Radiolabeled proteins from lysates of NIH 3T3 cells, either mock infected or infected with WT MCMV (1 PFU/cell), were immunoprecipitated with anti-M141 serum (first immunoprecipitation). The immune (more ...) Complex formation in the absence of virus infection.
In order to assess whether these viral proteins formed stable complexes in the absence of other viral proteins (or cellular proteins induced by virus infection), we repeated the immunoprecipitation experiments by using lysates from fibroblasts transiently expressing M139, M140, and M141. For these experiments we constructed plasmids expressing epitope-tagged versions of the viral proteins to completely avoid any antibody cross-reactivity and to facilitate detection of the viral proteins by Western blotting. Initially, NIH 3T3 cells were cotransfected with vectors expressing His/Xpress-tagged M139, FLAG-tagged M140, and either FLAG-tagged M141 or, as a control, FBAP. The anti-His antibody detects only the 75-kDa form of the His/Xpress-tagged M139 protein, in contrast to the polyclonal anti-M139 serum, which detects both the 75- and 61-kDa forms. In addition, in these initial experiments, the FLAG-tagged M140 (truncated by 81 amino acids) and FLAG-tagged M141 proteins were of similar sizes, thus preventing their distinction based on molecular weight but nonetheless providing epitope-tagged binding partners for tagged p75M139.
The data in Fig. confirmed that the viral gene products were expressed as expected in the transfected fibroblasts. The p75M139 protein was expressed abundantly in samples cotransfected with His/Xpress-tagged-p75M139 and FLAG-tagged M140 (lane 2). This product was detected at much lower, but detectable, levels in cells transfected with all three US22 genes (lane 3). The FLAG-tagged viral proteins were detected in cells transfected with FLAG-tagged M140- and M141-expressing vectors (lanes 2 and 3). After immunoprecipitation of the transfected-cell lysates with either anti-M139 or anti-His serum, coprecipitating FLAG-tagged M140 and M141 were identified by Western blotting (Fig. ). Importantly, FLAG-tagged M140 did not coprecipitate with His/Xpress-M139 in the absence of M141 (lane 2). Only when all three viral genes were expressed did FLAG-M140 and FLAG-M141 coprecipitate with His/Xpress-M139 (lane 3). Identical results were seen when using anti-M139 or anti-His serum as the precipitating antibody. These data substantiate our hypothesis that the 75-kDa M139-derived protein binds to a pM140/pM141 complex in a conformation-dependent manner but not to the individual proteins. The results corroborate the data in Fig. , which showed that pM140 or pM141 did not coprecipitate with anti-M139 serum from lysates of cells infected with MCMV having M141 or M140 deleted, respectively.
FIG. 4. Products of M139, M140, and M141 form a complex when the genes are transiently expressed in the absence of other viral proteins. NIH 3T3 cells were cotransfected with vectors expressing His/Xpress-tagged M139 (H139), FLAG-tagged M140 (F140), and either (more ...)
A similar approach was used to further assess complexing of pM140 and pM141 in transiently transfected cells. In these experiments, vectors expressing either WT or His/Xpress-tagged-M140 were cotransfected with a vector expressing FLAG-tagged M141 or the FBAP control. The data in Fig. indicate that the FLAG-tagged proteins were expressed to comparable levels in the transfected cells. Importantly, when the cell lysates were immunoprecipitated with anti-M140 antiserum, coprecipitation of FLAG-tagged M141 with either the WT or His/Xpress-tagged M140 was evident by Western blotting (Fig. ). Thus, these two proteins form a stable complex in the absence of other viral proteins or cellular proteins induced by viral infection.
FIG. 5. Products of M140 and M141 form a complex, independent of M139, when the two genes are transiently expressed in the absence of other viral proteins. NIH 3T3 cells were transiently transfected with vectors expressing FLAG-tagged M141 (F141) and either His/Xpress-tagged (more ...) Complex formation in vitro.
In order to determine if cellular proteins are required to form or stabilize the complex of M139, M140, and M141 proteins, we assessed the ability of the three proteins to coimmunoprecipitate as products of in vitro transcription-translation reactions. When full-length M139 and M141 were cotranscribed and translated along with His/Xpress-tagged full-length M140 in a single reaction, all three products were expressed to nearly comparable levels (Fig. , first lane). The exception was the 61-kDa product of M139, which was not expressed to detectable levels. Immunoprecipitation with anti-His antibody yielded trace amounts of p75M139 and more abundant levels of pM140 and pM141 (Fig. , third lane). The differences in the intensities of p75M139 compared to the other two proteins may reflect detection by anti-His of a monomeric form of pM140 or a pM140/pM141 complex in addition to a complex composed of all three proteins; thus “pulling down” more pM140 and pM141 than p75M139. Importantly, these data demonstrate that complexes composed of M139, M140, and M141 gene products can form in the absence of cellular proteins. We cannot rule out the possibility, however, that the stoichiometry and/or stability of the complex may be influenced by cellular proteins not present in the translation reaction.
FIG. 6. Proteins p75M139, pM140, and pM141 form a complex when expressed as products of in vitro transcription and translation. Genes M139, His/Xpress-tagged M140, and M141 were cotranscribed and translated in vitro in the presence of [35S]methionine as described (more ...) Sedimentation gradient analyses to reveal the identity of the two complexes.
The experiments demonstrating coimmunoprecipitation of products of M139, M140, and M141 from infected cells, transfected cells, or in vitro-transcribed genes provided strong evidence that these proteins complex and that at least two separate complexes exist under steady-state conditions. To further prove the physical identity of these complexes, lysates of cells infected with WT virus or RVΔ140 were subjected to sedimentation sucrose gradient analyses, and the various fractions were analyzed for the presence of M139, M140, and M141 proteins. Although the data with fibroblasts are presented for consistency, results were identical using IC-21 macrophages as hosts for infection.
Initial experiments were designed to determine where the monomeric forms of these proteins, expressed in the absence of other binding partners, sedimented within the gradient. Products of each single gene transcribed and translated in vitro were applied to the gradient, and the fractions in which they sedimented were detected by autoradiography. As shown in Fig. , three distinct peaks were evident, representing each of the indicated proteins. The peak fraction for the M139 gene product represents p75M139, as p61M139 was not evident from the autoradiograph, similar to previous results of in vitro transcription-translation reactions (Fig. ). It is interesting that, in this analysis, the M141 product appeared to be of a greater size and/or weight than the product of M140. In spite of the faster migration of pM141 than of pM140 in SDS-polyacrylamide gels, a higher molecular weight for pM141 is actually predicted based on the amino acid sequence. The relative position of each of the proteins expressed as single genes was used to determine the identity of the “monomeric” forms of these proteins, although the presence of homodimers cannot be ruled out. However, upon consideration of the molecular weight of each protein on SDS-polyacrylamide gels, the relative positions of the three proteins within the gradient are suggestive of monomeric forms.
FIG. 7. Sucrose gradient fractionation of infected-cell lysates reveals three distinguishable complexes. (A) Fractionation of monomeric forms of each protein. Genes M139, M140, and M141 were individually transcribed and translated in vitro in the presence of (more ...)
Sedimentation of the three proteins in the context of virus infection was determined by subjecting lysates from infected fibroblasts or macrophages to gradient analyses and identifying the presence or absence of pM139 (p75M139 with or without p61M139), pM140, or pM141 in each fraction by Western dot blotting. As with conventional Western blotting, the polyclonal antibodies used to detect the viral proteins were not cross-reactive in these dot blot assays. A composite showing the relative sedimentation of each of the gene products from WT virus-infected cells is shown in Fig. .
The M139 protein(s) was identified in three separate fractions (Fig. ). Based on the data in Fig. , the peak of anti-M139 reactivity at fraction 11 corresponds to a monomeric form of this protein. As predicted from the immunoprecipitation assays, this monomeric form was also present in RVΔ140-infected cells. A second peak at fraction 15 was unpredicted but may correspond to a complex of p75M139 and/or p61M139 and an unidentified cellular or viral protein. Importantly, this peak was not present in cells infected with RVΔ140, in which pM140 is absent and pM141 is unstable under steady-state conditions (9
). A third anti-M139 reactive peak, detected in fraction 18 of WT virus-infected cells but not RVΔ140-infected cells, corresponds to the peak of pM140 and pM141 reactivity (Fig. ), thus confirming the existence of a complex composed of all three gene products.
In Fig. , a distinct monomeric form of pM140 was not evident; however, the broad left shoulder of the first anti-M140 reactive peak may mask a monomeric form at fraction 4. Two distinct pM140-containing peaks were evident in WT virus-infected cells, at fractions 9 and 18. Importantly, these same peaks correspond to pM141-containing fractions (Fig. ). The first peak represents the pM140/pM141 complex. The second corresponds to the complex of all three gene products. Whether the unknown 98-kDa protein identified in the initial coimmunoprecipitation experiments (Fig. ) is a component of one of these fractions has yet to be determined.
Finally, a monomeric form of pM141 was evident in fractions 6 to 7 (Fig. ), in addition to the two peaks mentioned above containing both pM140 and pM141 (Fig. ). In fractions of RVΔ140-infected cells, a small peak that may represent a monomeric form of pM141 was seen; however, considering the instability of this protein in the absence of pM140 under steady-state conditions, the true identify of this “peak” is in question.
Collectively, these data revealed the identify of three separate complexes composed of two or more of the M139, M140, and M141 gene products. As expected, one complex is composed of all three protein products (fraction 18) and a second (fraction 9) is composed of pM140 and pM141. A third complex (fraction 15) contains p75M139 and/or p61M139 and one or more as-yet-unidentified protein(s) either encoded by M140 or regulated by the product of this gene. Significantly, these experiments revealed structural identities to which function can eventually be ascribed.
Colocalization of M139, M140, and M141 proteins to a perinuclear region of the cell.
The above evidence demonstrates the presence of at least three complexes composed of the M139, M140, and M141 proteins in various combinations. Previous studies indicated that each gene product was detected, via cell fractionation and Western blotting, in both the nucleus and cytoplasm of MCMV-infected cells (9
). To identify the cellular region where the M139, M140, and M141 gene products converge and, hence, where the complexes might function within infected cells, we performed confocal microscopy analyses. Here we made use of cells transiently expressing epitope-tagged products.
The confocal images in Fig. indicate that, when coexpressed, products from all three genes colocalize predominantly to a perinuclear region of the cell. Based on the Western blot data above, we assume that the majority of the FITC staining of His/Xpress-tagged M139 is due to expression of p75M139. This product was expressed predominately in the perinuclear region of the cytoplasm, although some nuclear staining, in discrete regions, was observed as expected. FLAG-tagged pM140 and pM141 also localized to the perinuclear region of the cytoplasm, with faint nuclear staining. Merged images revealed perinuclear regions where the M139, M140, and M141 products converged. This area of colocalization likely reflects the site where the M139-M140-M141 complex resides.
FIG. 8. Products of M139, M140, and M141 colocalize to a perinuclear region of the cell juxtaposed to or within the cis-Golgi region. (A) NIH 3T3 cells were cotransfected with His/Xpress-tagged M139 (His-M139), FLAG-tagged M140 (FLAG-M140), and FLAG-tagged M141 (more ...)
As expression and localization of FLAG-tagged M140 and FLAG-tagged M141 could not be distinguished in Fig. , we performed additional experiments to determine the specific localization of each of these two proteins. In these experiments, FLAG-tagged M140 expressed in the absence of pM141 resided predominantly within the nucleus, with faint cytoplasmic staining (Fig. ). This finding was somewhat surprising, as the sequence of M140 does not reveal a consensus nuclear localization signal. In contrast, Myc-tagged pM141 expressed alone displayed a diffuse cytoplasmic staining, with some nuclear localization. However, when pM140 and pM141 were coexpressed, they colocalized to the perinuclear region in a punctate pattern of staining, with some pM140 remaining within the nucleus. These data suggest that coexpression of pM140 and pM141, under conditions where the two proteins complex, alters the predominant subcellular localization of each protein. Presumably, the physical interaction of pM140 with pM141 either prevents pM140 targeting to the nucleus or retrieves pM140 from the nucleus. Furthermore, these data indicate that the free forms of these proteins reside in cellular regions different from the pM140/pM141 complex or the pM139/pM140/pM141 complex.
We next performed experiments to determine if the transition of pM140 from a nuclear to a perinuclear region upon expression of pM141 was evident within the environment of an MCMV-infected cell. Therefore, fibroblasts transfected with GFP-tagged M140 were superinfected with MCMV mutants having M140 deleted and either expressing M141 or having M141 deleted. The images in Fig. indicate that, in the absence of pM141, when cells were either mock infected or infected with mutant MCMV having M139, M140, and M141 deleted (RV10), pM140 remained largely nuclear. However, when the GFP-M140-expressing cells were superinfected with MCMV expressing M141 but having either M139 and M140 deleted (RV12) or M140 alone deleted (RV14), pM140 was expressed predominantly in the cytoplasm in discrete, punctate patterns. Although precise subcellular localization of the punctate staining is more difficult to interpret in live cells with virus-induced cytopathic effects, the differences in the staining pattern of pM140 in the absence versus the presence of pM141 confirmed that pM141 influences the localization of pM140 within the context of MCMV infection. Results with RV12- compared to RV14-infected cells indicated that, as expected from results with transiently transfected cells, the influence of pM141 on pM140 localization was independent of M139 expression.
The particular perinuclear staining pattern of the M139, M140, and M141 products suggested that these proteins may converge in a Golgi-related compartment. Therefore, fibroblasts cotransfected with FLAG-tagged M140 and His-tagged M141 were stained with antibody to GS15 or GS28, components of the SNARE complex that mediates transport from the endoplasmic reticulum to the cis
-Golgi region and intra-Golgi transport (26
), or with FITC-labeled wheat germ agglutinin, which binds to sialic acid moieties in the lumen of the trans
-Golgi region. The data in Fig. indicate that pM140 and pM141 colocalize with GS15 and GS28, suggesting a cis
- or medial Golgi site of convergence. Cross-reactivity of anti-GS15 or -GS28 antibodies with MCMV proteins was ruled out (W. Salb, unpublished data). The complete absence of colocalization with wheat germ agglutinin provided evidence that pM140 and pM141 do not colocalize to the lumen of the trans
-Golgi region and apparently do not reside even proximal to this site, such as the cytoplasmic side of the trans
-Golgi region (data not shown). Thus, the data are consistent with localization of at least pM140 and pM141 to a region either closely juxtaposed to or within the cis
The majority of these experiments were performed using transfected fibroblasts, as the transfection efficiency of these cells is quite high. Selective experiments were repeated using IC-21 macrophages, which have a transfection efficiency of approximately 5%. The same results in colocalization of both the viral proteins and Golgi markers were seen in the macrophages (data not shown).