FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2161697105> ?p ?o ?g. }

• W2161697105 endingPage "262" @default.
• W2161697105 startingPage "253" @default.
• W2161697105 abstract "Human cytomegalovirus proteins alter host cells to favor virus replication. These viral proteins include pUL38, which prevents apoptosis. To characterize the mode of action of pUL38, we modified the viral genome to encode an epitope-tagged pUL38 and used rapid immunoaffinity purification to isolate pUL38-interacting host proteins, which were then identified by mass spectrometry. One of the cellular proteins identified was TSC2, a constituent of the tuberous sclerosis tumor suppressor protein complex (TSC1/2). TSC1/2 integrates stress signals and regulates the mammalian target of rapamycin complex 1 (mTORC1), a protein complex that responds to stress by limiting protein synthesis and cell growth. We showed that pUL38 interacts with TSC1 and TSC2 in cells infected with wild-type cytomegalovirus. Furthermore, TSC1/2 failed to regulate mTORC1 in cells expressing pUL38, and these cells exhibited the enlarged size characteristic of cytomegalovirus infection. Thus, pUL38 supports virus replication at least in part by blocking cellular responses to stress. Human cytomegalovirus proteins alter host cells to favor virus replication. These viral proteins include pUL38, which prevents apoptosis. To characterize the mode of action of pUL38, we modified the viral genome to encode an epitope-tagged pUL38 and used rapid immunoaffinity purification to isolate pUL38-interacting host proteins, which were then identified by mass spectrometry. One of the cellular proteins identified was TSC2, a constituent of the tuberous sclerosis tumor suppressor protein complex (TSC1/2). TSC1/2 integrates stress signals and regulates the mammalian target of rapamycin complex 1 (mTORC1), a protein complex that responds to stress by limiting protein synthesis and cell growth. We showed that pUL38 interacts with TSC1 and TSC2 in cells infected with wild-type cytomegalovirus. Furthermore, TSC1/2 failed to regulate mTORC1 in cells expressing pUL38, and these cells exhibited the enlarged size characteristic of cytomegalovirus infection. Thus, pUL38 supports virus replication at least in part by blocking cellular responses to stress. Human cytomegalovirus (HCMV) is a member of the β-herpesvirus family. Infections in healthy children and adults are generally asymptomatic, but the virus causes life-threatening disease in immunologically immature or compromised individuals (reviewed in Mocarski et al., 2007Mocarski E.S. Shenk T. Pass R.F. Cytomegaloviruses.in: Knipe D.M. Howley P.M. Fields Virology. Lippincott, Williams and Wilkins, Philadelphia, PA2007: 2702-2772Google Scholar). Congenital HCMV infection is the leading viral cause of birth defects, and neonates can suffer serious complications following infection. HCMV is a major complication in immunosuppressed individuals, with a significant contribution to morbidity and mortality in allogeneic transplant recipients and AIDS patients. The HCMV genome contains >200 open reading frames, although many have not been demonstrated to encode proteins (Murphy et al., 2003aMurphy E. Rigoutsos I. Shibuya T. Shenk T.E. Reevaluation of human cytomegalovirus coding potential.Proc. Natl. Acad. Sci. USA. 2003; 100: 13585-13590Crossref PubMed Scopus (142) Google Scholar, Murphy et al., 2003bMurphy E. Yu D. Grimwood J. Schmutz J. Dickson M. Jarvis M.A. Hahn G. Nelson J.A. Myers R.M. Shenk T.E. Coding potential of laboratory and clinical strains of human cytomegalovirus.Proc. Natl. Acad. Sci. USA. 2003; 100: 14976-14981Crossref PubMed Scopus (395) Google Scholar). Upon infection of a permissive cell, HCMV expresses its genes in a regulated cascade; immediate-early genes are expressed first, followed by early and then late genes. The UL38 transcription unit is first expressed during the early phase of infection (reviewed in Mocarski et al., 2007Mocarski E.S. Shenk T. Pass R.F. Cytomegaloviruses.in: Knipe D.M. Howley P.M. Fields Virology. Lippincott, Williams and Wilkins, Philadelphia, PA2007: 2702-2772Google Scholar. A mutant virus lacking pUL38 induces apoptosis after infection, producing reduced levels of viral progeny (Terhune et al., 2007Terhune S. Torigoi E. Moorman N. Silva M. Qian Z. Shenk T. Yu D. Human cytomegalovirus UL38 protein blocks apoptosis.J. Virol. 2007; 81: 3109-3123Crossref PubMed Scopus (116) Google Scholar). The mechanism by which pUL38 blocks apoptosis and facilitates HCMV growth is unknown. A BLAST search of pUL38 reveals no sequence homology to cellular proteins, and more sophisticated searches for functional homologies also failed to provide compelling hints to its mode of action (Novotny et al., 2001Novotny J. Rigoutsos I. Coleman D. Shenk T. In silico structural and functional analysis of the human cytomegalovirus (HHV5) genome.J. Mol. Biol. 2001; 310: 1151-1166Crossref PubMed Scopus (40) Google Scholar, Rigoutsos et al., 2003Rigoutsos I. Novotny J. Huynh T. Chin-Bow S.T. Parida L. Platt D. Coleman D. Shenk T. In silico pattern-based analysis of the human cytomegalovirus genome.J. Virol. 2003; 77: 4326-4344Crossref PubMed Scopus (51) Google Scholar). To probe the role of pUL38, we screened for proteins that interact with it. We used a mutant virus, expressing an epitope-tagged pUL38 protein from its normal context in the viral genome, coupled with a rapid one-step immunoaffinity purification and mass spectrometry to identify interacting proteins. This combination of proteomics and genetics identified multiple viral and cellular proteins likely to interact with pUL38, one of which is TSC2, also known as tuberin. TSC2 and TSC1 (hamartin) interact to form the tuberous sclerosis protein complex (TSC1/2), and mutations in either subunit are linked to the development of tuberous sclerosis, a recessive disorder that is characterized by tumors in multiple organs (reviewed in Crino et al., 2006Crino P.B. Nathanson K.L. Henske E.P. The tuberous sclerosis complex.N. Engl. J. Med. 2006; 355: 1345-1356Crossref PubMed Scopus (1204) Google Scholar). TSC1/2 is regulated by multiple signaling pathways (reviewed in Kwiatkowski and Manning, 2005Kwiatkowski D.J. Manning B.D. Tuberous sclerosis: A GAP at the crossroads of multiple signaling pathways.Hum Mol Genet. 2005; 14: R251-R258Crossref PubMed Scopus (316) Google Scholar). Growth factors activate Akt and RSK1, which phosphorylate TSC2 and block its activity. Stress activates AMP kinase (AMPK), which phosphorylates TSC2 and activates it. When the TSC1/2 complex is activated, TSC2 functions as a GTPase-activating protein for Rheb, a GTP-binding protein that activates the mammalian target of rapamycin complex 1 (mTORC1). mTORC1 is comprised of at least three subunits, mTOR serine-threonine kinase, raptor, and gβL, and it regulates cell growth in response to growth factors and nutrient availability. mTORC1 controls cell growth by modulating multiple processes, including protein synthesis, ribosome biogenesis, and autophagy (reviewed in Sarbassov et al., 2005aSarbassov D.D. Ali S.M. Sabatini D.M. Growing roles for the mTOR pathway.Curr. Opin. Cell Biol. 2005; 17: 596-603Crossref PubMed Scopus (1237) Google Scholar). Thus, TSC1/2 interprets signals from multiple inputs, and when activated, it is a negative regulator of mTORC1 and thereby inhibits cellular growth. We confirmed the interaction of pUL38 with the tumor suppressor protein complex and demonstrated that the viral protein antagonizes the ability of TSC1/2 to negatively regulate mTORC1. Thus, pUL38 blocks a growth regulatory pathway to facilitate viral replication. To identify cellular and viral proteins that interact with pUL38 in the context of infection, we created a viral mutant, BADinUL38TAP, which expresses from its normal location on the viral genome the pUL38 protein fused to immunoglobulin-binding domains of protein A and a calmodulin-binding peptide (TAP) at its carboxyl terminus, pUL38TAP (Figure 1A, left). Mutations disrupting the UL38 ORF result in attenuated virus replication with a rapid onset of apoptosis (Terhune et al., 2007Terhune S. Torigoi E. Moorman N. Silva M. Qian Z. Shenk T. Yu D. Human cytomegalovirus UL38 protein blocks apoptosis.J. Virol. 2007; 81: 3109-3123Crossref PubMed Scopus (116) Google Scholar). BADinUL38TAP replicated to wild-type levels (Figure 1A, right), and pUL38TAP displayed a similar localization as observed for untagged pUL38 when assayed by immunofluorescence (Figure 1B). Tagged pUL38 (from BADinUL38TAP) and untagged pUL38 (from BADWT) were found in both the cytoplasm and nucleus at 24 hr after infection. The pUL38 TAP fusion does not disrupt the localization or an essential role of pUL38 in HCMV-infected fibroblasts. At 24 hr postinfection with BADinUL38TAP, pUL38TAP and associated proteins were isolated from cell extracts. By capturing complexes from cells infected with the HCMV variant, it was possible to identify pUL38-interacting proteins under conditions where pUL38 is expressed with proper kinetics and at normal levels. Isolations were performed via the protein A tag, using a rapid one-step immunoaffinity purification on magnetic beads coated with IgG (Cristea et al., 2005Cristea I.M. Williams R. Chait B.T. Rout M.P. Fluorescent proteins as proteomic probes.Mol. Cell. Proteomics. 2005; 4: 1933-1941Crossref PubMed Scopus (195) Google Scholar, Cristea et al., 2006Cristea I.M. Carroll J.W. Rout M.P. Rice C.M. Chait B.T. Macdonald M.R. Tracking and elucidating alphavirus-host protein interactions.J. Biol. Chem. 2006; 281: 30269-30278Crossref PubMed Scopus (141) Google Scholar). pUL38TAP was efficiently captured with little of the fusion protein remaining in the insoluble fraction (data not shown). Isolated viral and host proteins were resolved by electrophoresis, stained with Coomassie blue, and identified by mass spectrometry. The major protein bands evident in the polyacrylamide gel and illustrative data from sequential MALDI QqTOF MS and MALDI IT MS/MS analysis for one of the identified cell proteins, TSC2, are displayed in Figure 1C. The full set of identified proteins is presented in Table S1 (available online). Two HCMV proteins were identified in the capture experiment (Table S1). The first was pUL52, and the second migrated at ∼70 kDa (Figure 1C). The latter contained amino acid segments from the adjacent UL29 and UL28 ORFs. NetGene2 (http://www.cbs.dtu.dk/services/NetGene2/; Brunak et al., 1991Brunak S. Engelbrecht J. Knudsen S. Prediction of human mRNA donor and acceptor sites from the DNA sequence.J. Mol. Biol. 1991; 220: 49-65Crossref PubMed Scopus (598) Google Scholar) predicted a splice donor/acceptor motif that would generate a mRNA encoding a UL29/28 protein of 701 amino acids, consistent with its electrophoretic migration. The product of UL52 is essential, while the UL29 and 28 ORFs augment HCMV replication in fibroblasts (Yu et al., 2003Yu D. Silva M.C. Shenk T. Functional map of human cytomegalovirus AD169 defined by global mutational analysis.Proc. Natl. Acad. Sci. USA. 2003; 100: 12396-12401Crossref PubMed Scopus (314) Google Scholar). However, their functions are unknown, and we have not investigated the consequences of their predicted interactions with pUL38. Numerous cellular proteins were identified in the capture experiment (Figure 1C and Table S1). Although their capture suggests that pUL38 might influence multiple cell functions, we have so far confirmed only one of these interactions. The list, therefore, comprises potential pUL38-interacting proteins. It is intriguing that six subunits of the nucleosome remodeling and histone deacetylation (NuRD) complex were among the proteins captured by pUL38TAP: Mi-2β, MTA1 and 2, HDAC1 and 2, and RbAp48/46 (Figure 1C). The NuRD complex includes histone deacetylases and chromatin-remodeling ATPases, which repress transcription (Bowen et al., 2004Bowen N.J. Fujita N. Kajita M. Wade P.A. Mi-2/NuRD: Multiple complexes for many purposes.Biochim. Biophys. Acta. 2004; 1677: 52-57Crossref PubMed Scopus (243) Google Scholar). It is possible that pUL38 antagonizes NuRD to optimize expression of the viral genome. The HCMV immediate-early 1 (Nevels et al., 2004Nevels M. Paulus C. Shenk T. Human cytomegalovirus immediate-early 1 protein facilitates viral replication by antagonizing histone deacetylation.Proc. Natl. Acad. Sci. USA. 2004; 101: 17234-17239Crossref PubMed Scopus (147) Google Scholar) and immediate-early 2 (Park et al., 2007Park J.J. Kim Y.E. Pham H.T. Kim E.T. Chung Y.H. Ahn J.H. Functional interaction of the human cytomegalovirus IE2 protein with histone deacetylase 2 in infected human fibroblasts.J. Gen. Virol. 2007; 88: 3214-3223Crossref PubMed Scopus (48) Google Scholar) proteins also block histone deacetylase function, and other herpesviruses (e.g., Gu et al., 2005Gu H. Liang Y. Mandel G. Roizman B. Components of the REST/CoREST/histone deacetylase repressor complex are disrupted, modified, and translocated in HSV-1-infected cells.Proc. Natl. Acad. Sci. USA. 2005; 102: 7571-7576Crossref PubMed Scopus (156) Google Scholar) attack repressive chromatin-modifying complexes as well. We focused on the predicted interaction of pUL38 with TSC2, a component of the TSC1/2 tumor suppressor protein complex. TSC1/2 regulates mTORC1, which is deregulated by HCMV infection (Kudchodkar et al., 2004Kudchodkar S.B. Yu Y. Maguire T.G. Alwine J.C. Human cytomegalovirus infection induces rapamycin-insensitive phosphorylation of downstream effectors of mTOR kinase.J. Virol. 2004; 78: 11030-11039Crossref PubMed Scopus (128) Google Scholar). This led to the hypothesis that pUL38 binds to TSC1/2 and antagonizes its ability to regulate mTORC1. To confirm the putative pUL38-TSC2 interaction, we reversed the capture process used in the pUL38TAP immunoaffinity purification, and used antibodies specific for cellular proteins to test for coimmunoprecipitation of pUL38 from wild-type virus-infected cell extracts. A TSC2-specific antibody coprecipitated pUL38 from infected cells, but not mock-infected cells (Figure 2A, top panel). No pUL38 was detected after immunoprecipitation from the infected cell extract with preimmune IgG, and the use of wild-type virus ruled out a nonspecific interaction of TSC2 with the TAP component of pUL38TAP. The thickness of the pUL38 band detected in Figure 2A suggested that multiple species might be present, so the analysis was repeated using a higher-resolution electrophoresis protocol (Figure 2B). Three pUL38-specific bands were evident, corresponding to proteins of approximately 33, 35, and 37 kDa. All three isoforms are found in cells expressing only pUL38 (Figure 4A), indicating that the three species are specific to the UL38 ORF. We do not yet know the origin of the three species but note that there are three in-frame AUG codons that could code for proteins this size, and there is precedent in HCMV for use of multiple in-frame starts within an ORF (Stamminger et al., 2002Stamminger T. Gstaiger M. Weinzierl K. Lorz K. Winkler M. Schaffner W. Open reading frame UL26 of human cytomegalovirus encodes a novel tegument protein that contains a strong transcriptional activation domain.J. Virol. 2002; 76: 4836-4847Crossref PubMed Scopus (57) Google Scholar). Antibody to TSC2 preferentially coprecipitated the pUL38 37 kDa isoform and to a lesser extent the 35 kDa species (Figure 2B). TSC2 interacts with TSC1 to form the tumor suppressor protein complex TSC1/2. To determine whether pUL38 also interacts, directly or indirectly, with TSC1, the same set of lysates examined in Figure 2A were subjected to immunoprecipitation with antibody to pUL38 (Figure 2C, top panel). pUL38-specific immune complexes isolated from BADWT-infected cells contained TSC1 protein, and this interaction was found to be specific using the same criteria outlined above for TSC2. A similar experiment demonstrated that antibody to TSC1 can coprecipitate pUL38 (Figure 2D). To determine whether pUL38 can interact with each of the TSC1/2 subunits independently, 293T cells were transfected with a pUL38 expression vector plus constructs encoding FLAG-tagged TSC1 and/or FLAG-tagged TSC2. pUL38 was coprecipitated with tagged TSC2 but not TSC1 (Figure 2E), arguing that the viral protein does not interact with free TSC1. To further probe the interaction of the viral protein with TSC2, cells were transfected with the pUL38 expression vector plus a vector encoding GFP-tagged TSC2 or a GFP-tagged derivative of TSC2 lacking the TSC1 interaction domain (Goncharova et al., 2004Goncharova E. Goncharov D. Noonan D. Krymskaya V.P. TSC2 modulates actin cytoskeleton and focal adhesion through TSC1-binding domain and the Rac1 GTPase.J. Cell Biol. 2004; 167: 1171-1182Crossref PubMed Scopus (88) Google Scholar). The deleted TSC2 was, as expected, smaller than the wild-type protein, and immunoprecipitation of the TSC2 variant with GFP-specific antibody coprecipitated pUL38 (Figure 2F). We conclude that pUL38 interacts with the tumor suppressor complex primarily through its TSC2 subunit. A direct interaction with TSC2, but not TSC1, might explain the failure to detect TSC1 in our analysis of pUL38TAP-interacting proteins by mass spectrometry (Table S1). Perhaps the TSC1/2 complex is disrupted during the one-step isolation method. Since pUL38 can interact with a TSC2 variant lacking a TSC1-binding domain, it is likely that pUL38 does not disrupt the TSC1/2 complex. To verify this prediction, we tested whether normal levels of the TSC1/2 complex were maintained in infected cells (Figure 2G). Cell lysates were prepared after mock or BADWT infection and subjected to immunoprecipitation with antibody to TSC2, and coprecipitated TSC1 was monitored by western blot assay. TSC1 was present in TSC2 immune precipitates at each time assayed after infection. In fact, more TSC1 was found associated with TSC2 at 72 and 96 hpi, consistent with the increase observed in the total amount of TSC1; in contrast, the level of TSC2 remained relatively constant after infection. In a recent high-throughput analysis, TSC1 protein was shown to increase by a factor of 3.1 after HCMV infection (Stanton et al., 2007Stanton R.J. McSharry B.P. Rickards C.R. Wang E.C. Tomasec P. Wilkinson G.W. Cytomegalovirus destruction of focal adhesions revealed in a high-throughput Western blot analysis of cellular protein expression.J. Virol. 2007; 81: 7860-7872Crossref PubMed Scopus (38) Google Scholar). To further investigate the interaction of pUL38 with the TSC1/2 complex, we performed immunofluorescent analysis. Visual inspection of the fluorescent images suggested that TSC1 and TSC2 exhibited substantial colocalization within uninfected and infected cells (Figure 3), and quantitative measurement of the images (Costes et al., 2004Costes S.V. Daelemans D. Cho E.H. Dobbin Z. Pavlakis G. Lockett S. Automatic and quantitative measurement of protein-protein colocalization in live cells.Biophys. J. 2004; 86: 3993-4003Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar) confirmed the colocalization, demonstrating that Pearson's correlation (r) for colocalization was even greater in infected (r = 0.79) than uninfected cells (r = 0.65). This difference was consistently observed in multiple images (data not shown). Perfect colocalization was evident when a TSC1 fluorescent image was compared to itself (r = 1.0), and little was evident when cytoplasmic virus-coded pUL99 protein was compared to DAPI-stained DNA (r = 0.06). We infer that pUL38 does not significantly disrupt the normal association of TSC1 and TSC2. Since TSC1/2 normally inhibits the mTORC1 kinase under stress conditions, limiting cell size and mass (Sarbassov et al., 2005aSarbassov D.D. Ali S.M. Sabatini D.M. Growing roles for the mTOR pathway.Curr. Opin. Cell Biol. 2005; 17: 596-603Crossref PubMed Scopus (1237) Google Scholar), we tested whether pUL38 can release this constraint. Fibroblasts were generated expressing pUL38 (HFF-pUL38) or GFP (HFF-GFP). Initially, we compared pUL38 expression in HFF-pUL38 cells to that in fibroblasts at 48 hr after infection with BADWT (Figure 4A). Western blot analysis demonstrated that similar amounts of pUL38 and the same variety and relative proportions of pUL38 subspecies were produced in cells expressing the protein as in infected cells. Further, antibody to TSC2 coimmunoprecipitated pUL38 from extracts of HFF-pUL38 cells (Figure 4B, top panel), demonstrating that no additional virus-coded protein is needed for the interaction. TSC2-specific antibody also coimmunoprecipitated TSC1 (Figure 4B, second panel from top), consistent with our interpretation that the TSC1/2 complex remains intact in the presence of pUL38. Western blot assays demonstrated that extracts of HFF-pUL38 and HFF-GFP cells contained very similar amounts of TSC1 and TSC2 (Figure 4B, bottom panels). The failure to modulate TSC1 levels in the presence of pUL38 suggests that one or more additional virus-coded functions is needed for the induction observed in infected cells (Figure 2). Cell volumes were assayed by measurement of calcein green AM fluorescence in sequential 0.3 μm optical sections through cells, and the average volume calculated for HFF-pUL38 cells was about twice that of HFF-GFP cells (Figure 4C). Further, measurement of forward scatter by flow cytometry confirmed that HFF-pUL38 cells are larger than HFF-GFP cells (Figure 4D). Thus, pUL38-expressing cells were larger than control cells, consistent with the inhibition of TSC1/2 (Fingar et al., 2002Fingar D.C. Salama S. Tsou C. Harlow E. Blenis J. Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E.Genes Dev. 2002; 16: 1472-1487Crossref PubMed Scopus (802) Google Scholar). As noted above, TSC1/2 inhibits the activity of mTORC1 in response to stress. TSC2 is activated when serum is withdrawn from cells, because the loss of growth factors inhibits PI3K-Akt and ERK1/2-RSK1 signaling, which normally block TSC1/2 activity; TSC2 is also activated by the withdrawal of nutrients, because energy deprivation activates AMPK, which then activates TSC1/2 (Figure 7) (Kwiatkowski and Manning, 2005Kwiatkowski D.J. Manning B.D. Tuberous sclerosis: A GAP at the crossroads of multiple signaling pathways.Hum Mol Genet. 2005; 14: R251-R258Crossref PubMed Scopus (316) Google Scholar). When activated, mTORC1 phosphorylates the ribosomal protein S6 kinase (p70 S6 kinase) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1) (Sarbassov et al., 2005aSarbassov D.D. Ali S.M. Sabatini D.M. Growing roles for the mTOR pathway.Curr. Opin. Cell Biol. 2005; 17: 596-603Crossref PubMed Scopus (1237) Google Scholar). To further evaluate the ability of pUL38 to antagonize TSC1/2, we monitored the activity of mTORC1 in control or HFF-pUL38 cells after nutrient stress. Initially, the phosphorylation of rpS6 at S235/236, a target of the mTORC1-activated p70 S6 kinase, was assayed by using antibodies that recognized total or phosphorylated rpS6 (Figure 5A). Maintenance in medium lacking growth factors for 12 hr induced a modest decrease in total rpS6 in both cell types, and HFF-pUL38 cells accumulated ∼2.5-fold more phosphorylated rpS6 than HFF-GFP cells. Incubation in PBS (no growth factors, amino acids, or sugars) for 1 hr after the initial 12 hr period in medium lacking serum resulted in a dramatic reduction in the amount of phosphorylated rpS6 in HFF-GFP cells. In contrast, HFF-UL38 cells contained near wild-type levels of phosphorylated rpS6 after 1 hr in PBS, and the phosphoprotein was still detected, albeit at a reduced level, after 2 hr (Figure 5A). Continued phosphorylation of rpS6 in HFF-pUL38 cells after maintenance in PBS was dependent on rapamycin-sensitive mTORC1 activity (Figure 5B). We extended the analysis to direct targets of mTORC1 and found that phosphorylation of both p70 S6 kinase at T389 and 4E-BP1 at T37/46 was more resistant to a 2 hr incubation in PBS, following 12 hr in serum-free medium, in cells expressing pUL38 as compared to control cells (Figure 5B). Thus, pUL38 alone is sufficient to maintain mTORC1 signaling under stress-inducing conditions. Phosphorylation of 4E-BP1 T37/46 was more resistant to rapamycin in pUL38-expressing as compared to control cells (Figure 5B). The mechanistic basis for this observation is not clear, but it has been noted that HCMV-infected cells contain a rapamycin-resistant raptor-containing activity (raptor is a constituent of the normally rapamycin-sensitive mTORC1 complex) that can mediate hyperphosphorylation of 4E-BP1 (Kudchodkar et al., 2006Kudchodkar S.B. Yu Y. Maguire T.G. Alwine J.C. Human cytomegalovirus infection alters the substrate specificities and rapamycin sensitivities of raptor- and rictor-containing complexes.Proc. Natl. Acad. Sci. USA. 2006; 103: 14182-14187Crossref PubMed Scopus (86) Google Scholar). Nevertheless, rapamycin treatment at the start of infection delays the production of virus progeny by about 12 hr and reduces the final yield of virus by a factor of 5–50 in the presence or absence of serum (Kudchodkar et al., 2004Kudchodkar S.B. Yu Y. Maguire T.G. Alwine J.C. Human cytomegalovirus infection induces rapamycin-insensitive phosphorylation of downstream effectors of mTOR kinase.J. Virol. 2004; 78: 11030-11039Crossref PubMed Scopus (128) Google Scholar). Limiting nutrients induce an increase in the AMP/ATP ratio in the cell. AMP binds to and allows activation of AMPK, which can phosphorylate and activate TSC2 with subsequent inhibition of mTORC1 activity. Thus, AMPK negatively regulates mTORC1 through TSC1/2. AMPK also can be activated by the cell-permeable AMP analog AICAR. AICAR treatment decreases mTORC1 activity and induces cell growth arrest (Corton et al., 1995Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. 5-aminoimidazole-4-carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells?.Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (977) Google Scholar). We tested the ability of pUL38 to block the inhibition of mTORC1 activity by AMPK, using AICAR to stimulate AMPK activity. Serum-starved HFF-GFP control cells or HFF-pUL38 cells were treated with AICAR, and mTORC1 activity was assessed by measuring rpS6 phosphorylation at S235/236. Phosphorylated rpS6 was markedly reduced in HFF-GFP as compared to HFF-pUL38 cells after 3 hr or 6 hr of drug treatment (Figure 5C). The 6 hr AICAR treatment was repeated in the presence or absence of rapamycin (Figure 5D, top two panels). AICAR-induced phosphorylation of rpS6 was sensitive to the inhibitor, confirming that pUL38 preserved the activity of rapamycin-sensitive mTORC1. The concentration of AICAR used in these experiments induced phosphorylation at S79 of a known AMPK target, acetyl-CoA carboxylase (Figure 5E), demonstrating that AMPK was activated by the drug. Further, the level of phosphorylated acetyl-CoA carboxylase was not influenced by the presence of pUL38, arguing that pUL38 does not act at AMPK or upstream of AMPK to influence mTORC1 function. We conclude that pUL38 blocks the negative regulation of mTORC1 by AMPK by inhibiting TSC1/2 function. A second mTOR-containing complex, mTORC2, phosphorylates Akt at S473 (Hresko and Mueckler, 2005Hresko R.C. Mueckler M. mTOR.RICTOR is the Ser473 kinase for Akt/protein kinase B in 3T3-L1 adipocytes.J. Biol. Chem. 2005; 280: 40406-40416Crossref PubMed Scopus (485) Google Scholar, Sarbassov et al., 2005bSarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex.Science. 2005; 307: 1098-1101Crossref PubMed Scopus (4870) Google Scholar). This modification contributes to activation of Akt for efficient phosphorylation of some but not all of its targets (Guertin et al., 2006Guertin D.A. Stevens D.M. Thoreen C.C. Burds A.A. Kalaany N.Y. Moffat J. Brown M. Fitzgerald K.J. Sabatini D.M. Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1.Dev. Cell. 2006; 11: 859-871Abstract Full Text Full Text PDF PubMed Scopus (1043) Google Scholar, Jacinto et al., 2006Jacinto E. Facchinetti V. Liu D. Soto N. Wei S. Jung S.Y. Huang Q. Qin J. Su B. SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity.Cell. 2006; 127: 125-137Abstract Full Text Full Text PDF PubMed Scopus (1054) Google Scholar). Akt S473 phosphorylation is impaired in TSC1/2-deficient cells (Yang et al., 2006Yang Q. Inoki K. Kim E. Guan K.L. TSC1/TSC2 and Rheb have different effects on TORC1 and TORC2 activity.Proc. Natl. Acad. Sci. USA. 2006; 103: 6811-6816Crossref PubMed Scopus (135) Google Scholar), suggesting that the tumor suppressor regulates mTORC2, at least under some circumstances. Accordingly, we tested whether pUL38 influences Akt S473 phosphorylation as a consequence of its inhibitory interaction with TSC1/2. Although, as observed previously (Jacinto et al., 2006Jacinto E. Facchinetti V. Liu D. Soto N. Wei S. Jung S.Y. Huang Q. Qin J. Su B. SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity.Cell. 2006; 127: 125-137Abstract Full Text Full Text PDF PubMed Scopus (1054) Google Scholar), starvation in PBS blocked Akt S473 phosphorylation, there was no difference in the level of Akt S473 phosphorylation in HFF-pUL38 as compared to control HFF-GFP cells maintained in medium containing serum (Figure 5B, middle panels). We find no evidence for an effect of pUL38 on mTORC2. The effect of pUL38 on p70 S6 kinase and 4E-BP1 phosphorylation in response to stress was confirmed within HCMV-infected cells (Figure 6A). Fibroblasts were maintained in medium lacking serum overnight and then infected with BADWT (pUL38+) or BADdlUL38 (pUL38−). After virus adsorption, the inoculum was removed and replaced with serum-free medium, and the phosphorylation status of mTORC1 targets wa" @default.
• W2161697105 created "2016-06-24" @default.
• W2161697105 creator A5000668079 @default.
• W2161697105 creator A5034361556 @default.
• W2161697105 creator A5047437797 @default.
• W2161697105 creator A5047825308 @default.
• W2161697105 creator A5065548847 @default.
• W2161697105 creator A5084513610 @default.
• W2161697105 date "2008-04-01" @default.
• W2161697105 modified "2023-10-02" @default.
• W2161697105 title "Human Cytomegalovirus Protein UL38 Inhibits Host Cell Stress Responses by Antagonizing the Tuberous Sclerosis Protein Complex" @default.
• W2161697105 cites W1668086213 @default.
• W2161697105 cites W1965239527 @default.
• W2161697105 cites W1977225005 @default.
• W2161697105 cites W1981186210 @default.
• W2161697105 cites W1981803091 @default.
• W2161697105 cites W1982913109 @default.
• W2161697105 cites W1987105275 @default.
• W2161697105 cites W2000731858 @default.
• W2161697105 cites W2001259221 @default.
• W2161697105 cites W2002803921 @default.
• W2161697105 cites W2008460142 @default.
• W2161697105 cites W2011660868 @default.
• W2161697105 cites W2014181394 @default.
• W2161697105 cites W2016040396 @default.
• W2161697105 cites W2024460705 @default.
• W2161697105 cites W2030172852 @default.
• W2161697105 cites W2035394208 @default.
• W2161697105 cites W2037118986 @default.
• W2161697105 cites W2037166272 @default.
• W2161697105 cites W2038227076 @default.
• W2161697105 cites W2041573061 @default.
• W2161697105 cites W2042461504 @default.
• W2161697105 cites W2044293749 @default.
• W2161697105 cites W2046108122 @default.
• W2161697105 cites W2065447003 @default.
• W2161697105 cites W2082131764 @default.
• W2161697105 cites W2087812126 @default.
• W2161697105 cites W2088970299 @default.
• W2161697105 cites W2089119460 @default.
• W2161697105 cites W2093315880 @default.
• W2161697105 cites W2094904848 @default.
• W2161697105 cites W2096560689 @default.
• W2161697105 cites W2097000222 @default.
• W2161697105 cites W2098816998 @default.
• W2161697105 cites W2100002560 @default.
• W2161697105 cites W2100347100 @default.
• W2161697105 cites W2101182238 @default.
• W2161697105 cites W2108221845 @default.
• W2161697105 cites W2108360575 @default.
• W2161697105 cites W2109125050 @default.
• W2161697105 cites W2109263914 @default.
• W2161697105 cites W2110429239 @default.
• W2161697105 cites W2114858538 @default.
• W2161697105 cites W2117028091 @default.
• W2161697105 cites W2117827556 @default.
• W2161697105 cites W2120259492 @default.
• W2161697105 cites W2125782175 @default.
• W2161697105 cites W2132463840 @default.
• W2161697105 cites W2133594759 @default.
• W2161697105 cites W2135611431 @default.
• W2161697105 cites W2136913046 @default.
• W2161697105 cites W2137112042 @default.
• W2161697105 cites W2142084214 @default.
• W2161697105 cites W2146415848 @default.
• W2161697105 cites W2149012701 @default.
• W2161697105 cites W2157582689 @default.
• W2161697105 cites W2163583831 @default.
• W2161697105 cites W2170651190 @default.
• W2161697105 cites W2383259826 @default.
• W2161697105 cites W4238567307 @default.
• W2161697105 doi "https://doi.org/10.1016/j.chom.2008.03.002" @default.
• W2161697105 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2759192" @default.
• W2161697105 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18407068" @default.
• W2161697105 hasPublicationYear "2008" @default.
• W2161697105 type Work @default.
• W2161697105 sameAs 2161697105 @default.
• W2161697105 citedByCount "178" @default.
• W2161697105 countsByYear W21616971052012 @default.
• W2161697105 countsByYear W21616971052013 @default.
• W2161697105 countsByYear W21616971052014 @default.
• W2161697105 countsByYear W21616971052015 @default.
• W2161697105 countsByYear W21616971052016 @default.
• W2161697105 countsByYear W21616971052017 @default.
• W2161697105 countsByYear W21616971052018 @default.
• W2161697105 countsByYear W21616971052019 @default.
• W2161697105 countsByYear W21616971052020 @default.
• W2161697105 countsByYear W21616971052021 @default.
• W2161697105 countsByYear W21616971052022 @default.
• W2161697105 countsByYear W21616971052023 @default.
• W2161697105 crossrefType "journal-article" @default.
• W2161697105 hasAuthorship W2161697105A5000668079 @default.
• W2161697105 hasAuthorship W2161697105A5034361556 @default.
• W2161697105 hasAuthorship W2161697105A5047437797 @default.
• W2161697105 hasAuthorship W2161697105A5047825308 @default.
• W2161697105 hasAuthorship W2161697105A5065548847 @default.
• W2161697105 hasAuthorship W2161697105A5084513610 @default.
• W2161697105 hasBestOaLocation W21616971051 @default.

• next