A Mechanistic Study Of Human Cytomegalovirus PUL38 Activating MTORC1

Human cytomegalovirus(HCMV) is a member of the betaherpesvirus family with broad cell tropism. HCMV infection is a leading cause of birth defects and causes severe diseases in immunocompromised individuals. Following primary infection, HCMV is unable to be cleared by the host immune system, and persists as a lifelong latent and recurrent infection in the host. As an obligate cellular pathogen, HCMV has evolved multiple mechanisms allowing it to maintain a favorable cellular environment.The mammalian target of rapamycin complex 1(m TORC1), which plays a central role in the regulation of protein translation and anabolic metabolism, is a major target of virus manipulation. Viruses have evolved diverse mechanisms to activate this important cellular pathway by targeting m TORC1 or its up- or downstream components. HCMV maintains high m TORC1 activity despite the stressful cellular environment associated with infection. The multi-functional HCMV protein p UL38 has previously been reported to activate m TORC1 by binding to and antagonizing tuberous sclerosis complex protein 2(TSC2). p UL38 also plays a role in blocking endoplasmic reticulum stress-induced cell death during HCMV infection.In this study, we showed that a mutant p UL38 lacking the N-terminal 24 amino acids(p HA-UL3825-331) was fully functional in suppressing cell death during infection. Interestingly, p HA-UL3825-331 also lost the ability to interact with TSC2 but it retained the ability to activate m TORC1, although to a lesser extent than full-length p HA-UL38. Recombinant virus expressing p HA-UL3825-331 produced approximately 10-fold less progeny virus than the parental wild type virus, suggesting the importance of p UL38-TSC2 interaction in supporting efficient virus propagation. Site-directed mutational analysis identified a TQ motif at amino acid residues 23-24 as critical for p UL38 interaction with TSC2. Importantly, when overexpressed, the TQ/AA substitution mutant p HA-UL38 TQ/AA was capable of activating m TORC1 just like p HA-UL3825-331. We also created TSC2-null U373-MG cell lines by CRISPR genome editing and showed that p UL38 was capable of further increasing m TORC1 activity in TSC2-null cells. This study therefore identified the residues important for p UL38-TSC2 interaction and demonstrated that p UL38 can activate m TORC1 in both TSC2-dependent and-independent manners.