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.
IMPORTANCE: 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.