Tuberous Sclerosis Complex Protein 2 (TSC2)-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 multi-functional HCMV protein pUL38 has previously been reported to activate mTORC1 by binding to and antagonizing tuberous sclerosis complex protein 2 (TSC2). 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-UL38_25-331) was fully functional in suppressing cell death during infection. Interestingly, pHA-UL38_25-331 lost the ability to interact with TSC2 but it retained the ability to activate mTORC1, although to a lesser extent than full-length pHA-UL38. Recombinant virus expressing pHA-UL38_25-331 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 23-24 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-UL38_25-331. 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. This study therefore identified the residues important for pUL38-TSC2 interaction and demonstrated that pUL38 can activate mTORC1 in both TSC2-dependent and -independent manners.

Importance Human cytomegalovirus (HCMV), like other viruses, depends exclusively on its host cell to propagate. It has therefore developed methods to protect against host stress responses and to usurp cellular processes to complete its life cycle. Mammalian target of rapamycin complex 1 (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 tuberous sclerosis complex protein 2 (TSC2)-dependent and TSC2-independent manners. pUL38-independent mode of mTORC1 activation has also 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.