Low dNTP levels are necessary but may not be sufficient for lentiviral restriction by SAMHD1

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Results of this study argue against a requirement for additional myeloid-specific host factors for SAMHD1 function

Text by Sarah Welbourn

 

SAMHD1 is a host protein that was recently identified to be one of a growing list of lentiviral restriction factors that the cell uses to try to inhibit viral replication. The full details of the mechanism of action of SAMHD1 in viral restriction remain controversial. SAMHD1 is known to be an efficient dNTPase; however, whether lowering cellular dNTP levels is the sole mechanism of restriction used and whether or not this activity is even necessary required further investigation. Indeed, some studies had instead reported an essential nuclease function of the protein. In this paper, we used SAMHD1 mutants together with inhibitor treatment to modulate dNTP levels in cells and demonstrate that low dNTP levels do appear to be necessary for SAMHD1 restriction activity while providing further evidence that SAMHD1 may possess an additional dNTP-independent function that also contributes to lentiviral restriction.

 

We first wondered whether SAMHD1 might have dNTPase-independent function when both we and another group reported that a phosphomimetic of SAMHD1 was deficient in HIV-1 restriction but still had at least some dNTPase activity in vitro. A key prerequisite of our current study clearly was the ability to measure cellular dNTP levels. Once we successfully set up the assay to measure dNTP levels in cells we indeed confirmed that our SAMHD1 phosphomimetic was still able to decrease cellular dNTP levels similar to the WT protein without causing significant restriction to viral infection. We naturally therefore hypothesized that the controversial nuclease activity of SAMHD1 might be involved, but, surprisingly, despite numerous attempts we were unable to confirm that SAMHD1 possesses such an activity.

 

Having a reliable assay for measuring dNTP levels in cells enabled us to more systematically investigate the important role of low dNTP levels for SAMHD1 restriction. This is where we wanted to be able to modulate dNTP levels independent of SAMHD1. While dNTP levels remained high in permissive dividing cells even in the presence of SAMHD1, we found that blocking dNTP synthesis with a hydroxyurea inhibitor was able to decrease dATP levels in HeLa cells to similar levels in cells containing SAMHD1 or not. However, the treated cells that did contain SAMHD1 showed less viral infection indicating low dNTP levels appear to be necessary but not sufficient for SAMHD1 restriction of lentiviral infection.

 

Our study aimed at further delineating the mechanism of action of SAMHD1 in viral restriction; however, there is a lot more work to be done before its role is fully understood!

 

Welbourn blog Fig1

Image legend

Low dNTPs appear to be necessary but not sufficient for lentiviral restriction. In dividing HeLa cells (A, -HU), SAMHD1 is only slightly able to decrease dATP levels and shows no restriction of lentiviral infection (B). When hydroxyurea is used to block dNTP synthesis, dATP levels are reduced independent of SAMHD1 (A, +HU) yet WT SAMHD1 is able to exert an additional effect on viral infection (C). HD/RN is a SAMHD1 active site mutant, SIV-GFP ∆vpx is an SIV reporter virus that does not contain the SAMHD1-counteracting Vpx protein.

Introducing the authors

Authors_Strebel_Welbourne

Klaus Strebel is a Senior Investigator at the National Institute of Allergy and Infectious Diseases (NIAID). Sarah Welbourn is a Research Fellow at the NIAID.

About the research

Low dNTP levels are necessary but may not be sufficient for lentiviral restriction by SAMHD1

Sarah Welbourn, Klaus Strebel

Virology, Volume 488, 15 January 2016, Pages 271–277