Poster abstracts
Poster number 64 submitted by Jenna Marie Antonucci
SAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity
Jenna M. Antonucci (Center for Retrovirus Research, Department of Veterinary Biosciences; Department of Microbiology), Corine St. Gelais (Center for Retrovirus Research, Department of Veterinary Biosciences), Jacob S. Yount (Department of Microbial Infection and Immunity), Yong Xiong (Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT), Baek Kim (Department of Pediatrics, Center for Drug Discovery, Emory University School of Medicine), Li Wu (Center for Retrovirus Research, Department of Veterinary Biosciences; Department of Microbiology; Department of Microbial Infection and Immunity)
Abstract:
SAMHD1 restricts HIV-1 replication in non-dividing cells by degrading dNTPs to a level that limits efficient HIV-1 reverse transcription (RT). It’s been reported that SAMHD1 acts as an RNase and restricts HIV-1 replication in non-dividing cells through degradation of viral genomic RNA (gRNA), which challenged the established mechanism of HIV-1 restriction. To clarify these conflicting results, we independently generated stable U937 cell lines expressing SAMHD1 wild-type (WT) and mutants purported to specifically retain dNTPase (Q548A) or RNase (D137N) activities. Our results show WT SAMHD1 and the two mutants equally restricted HIV-1 infection and decreased dNTP levels in differentiated U937 cells. To determine whether SAMHD1 degrades HIV-1 gRNA, we measured HIV-1 gRNA levels in HIV-1-infected cells. We found similar levels of HIV-1 gRNA among the three SAMHD1-expressing cell lines compared to the vector control cells, indicating SAMHD1 does not degrade HIV-1 gRNA. Furthermore, we measured the levels of HIV-1 late RT products in infected cells and observed a significant decrease relative to vector control cells, suggesting SAMHD1 restricts HIV-1 infection at the level of RT. This correlates with the reduced dNTP pools measured in SAMHD1-expressing cell lines. Overexpression of SAMHD1 in virus producer cells didn’t affect HIV-1 Gag expression, viral release or infectivity, suggesting that SAMHD1 does not degrade HIV-1 mRNA. To clarify whether SAMHD1 has a nuclease activity, we measured the ability of stringently purified full-length recombinant SAMHD1 to degrade ssDNA and ssRNA in vitro. All SAMHD1 preparations maintained a robust dNTPase activity; however, only background nuclease activity was observed in some preparations, indicating that the inconsistency of RNase activity is likely due to contamination. Overall, our data indicate that Q548A and D137N mutants of SAMHD1 do not distinguish the dNTPase and RNase function, and that dNTP hydrolysis is the most likely mechanism of SAMHD1-mediated HIV-1 restriction in non-dividing cells.
Keywords: HIV-1, Retrovirus, SAMHD1