title: Tuberous Sclerosis Complex Protein 2-Independent Activation of mTORC1 by Human Cytomegalovirus pUL38.
abstract: The mammalian target of rapamycin complex 1 (mTORC1) controls cell growth and anabolic metabolism and is a critical host factor activated by human cytomegalovirus (HCMV) for successful infection. The multifunctional HCMV protein pUL38 previously has been reported to activate mTORC1 by binding to and antagonizing tuberous sclerosis complex protein 2 (TSC2) (J. N. Moorman et al., Cell Host Microbe 3:253-262, 2008, http://dx.doi.org/10.1016/j.chom.2008.03.002). pUL38 also plays a role in blocking endoplasmic reticulum stress-induced cell death during HCMV infection. In this study, we showed that a mutant pUL38 lacking the N-terminal 24 amino acids (pHA-UL3825-331) was fully functional in suppressing cell death during infection. Interestingly, pHA-UL3825-331 lost the ability to interact with TSC2 but retained the ability to activate mTORC1, although to a lesser extent than full-length pHA-UL38. Recombinant virus expressing pHA-UL3825-331 replicated with ∼10-fold less efficiency than the wild-type virus at a low multiplicity of infection (MOI), but it grew similarly well at a high MOI, suggesting an MOI-dependent importance of pUL38-TSC2 interaction in supporting virus propagation. Site-directed mutational analysis identified a TQ motif at amino acid residues 23 and 24 as critical for pUL38 interaction with TSC2. Importantly, when expressed in isolation, the TQ/AA substitution mutant pHA-UL38 TQ/AA was capable of activating mTORC1 just like pHA-UL3825-331. We also created TSC2-null U373-MG cell lines by CRISPR genome editing and showed that pUL38 was capable of further increasing mTORC1 activity in TSC2-null cells. Therefore, this study identified the residues important for pUL38-TSC2 interaction and demonstrated that pUL38 can activate mTORC1 in both TSC2-dependent and -independent manners.
  
  
  OBJECTIVE
  HCMV, like other viruses, depends exclusively on its host cell to propagate. Therefore, it has developed methods to protect against host stress responses and to usurp cellular processes to complete its life cycle. mTORC1 is believed to be important for virus replication, and HCMV maintains high mTORC1 activity despite the stressful cellular environment associated with infection. mTORC1 inhibitors suppressed HCMV replication in vitro and reduced the incidence of HCMV reactivation in transplant recipients. We demonstrated that mTORC1 was activated by HCMV protein pUL38 in both TSC2-dependent and TSC2-independent manners. The pUL38-independent mode of mTORC1 activation also has been reported. These novel findings suggest the evolution of sophisticated approaches whereby HCMV activates mTORC1, indicating its importance in the biology and pathogenesis of HCMV.
authors.affiliation: Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, China Unit of Herpesvirus and Molecular Virology, Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Xuhui District, Shanghai, China.
authors.forename: Yadan
authors.initials: Y
authors.lastname: Bai
authors.affiliation: Unit of Herpesvirus and Molecular Virology, Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Xuhui District, Shanghai, China.
authors.forename: Baoqin
authors.initials: B
authors.lastname: Xuan
authors.affiliation: Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, China.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Liu
authors.affiliation: Unit of Viral Hepatitis, Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Xuhui District, Shanghai, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhong
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA dong.yu@novartis.com zkqian@ips.ac.cn.
authors.forename: Dong
authors.initials: D
authors.lastname: Yu
authors.affiliation: Unit of Herpesvirus and Molecular Virology, Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Xuhui District, Shanghai, China dong.yu@novartis.com zkqian@ips.ac.cn.
authors.forename: Zhikang
authors.identifier: http://orcid.org/0000-0002-2046-4748
authors.initials: Z
authors.lastname: Qian
mesh_terms: D020816:Amino Acid Motifs; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003587:Cytomegalovirus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003586:Cytomegalovirus Infections / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D006801:Humans; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D036022:Capsid Proteins; D046912:Multiprotein Complexes; C000624653:TSC2 protein, human; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; C539756:UL38 protein, human herpesvirus-5; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases