Abstract
Sterile alpha motif (SAM) and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) acts as a restriction factor for several RNA and DNA viruses by limiting the intracellular pool of deoxynucleoside triphosphates. Here, we investigated the regulation of SAMHD1 expression during human cytomegalovirus (HCMV) infection. SAMHD1 knockdown using shRNA increased the activity of the viral UL99 late gene promoter in human fibroblasts by 7- to 9-fold, confirming its anti-HCMV activity. We also found that the level of SAMHD1 was initially increased by HCMV infection but decreased partly at the protein level at late stages of infection. SAMHD1 loss was not observed with UV-inactivated virus and required viral DNA replication. This reduction of SAMHD1 was effectively blocked by MLN4924, an inhibitor of the Cullin-RING-E3 ligase (CRL) complexes, but not by bafilomycin A1, an inhibitor of vacuolar-type H+-ATPase. Indirect immunofluorescence assays further supported the CRL-mediated SAMHD1 loss at late stages of virus infection. Knockdown of CUL2 and to a lesser extent CUL1 using siRNA stabilized SAMHD1 in normal fibroblasts and inhibited SAMHD1 loss during virus infection. Altogether, our results demonstrate that SAMHD1 inhibits the growth of HCMV, but HCMV causes degradation of SAMHD1 at late stages of viral infection through the CRL complexes.
Highlights
Cellular restriction factors play an important role in limiting the growth of a virus in a certain cell type
To confirm the anti-human cytomegalovirus (HCMV) activity of SAMHD1, SAMHD1depleted human fibroblasts (HFs) cells were produced by transduction with lentiviral vectors expressing Short Hairpin RNA (shRNA)
We infected control and SAMHD1-knockdown cells with recombinant HCMV (Towne strain) containing the UL99luciferase reporter gene, in which luciferase gene expression is driven by the viral UL99 late gene promoter, at a multiplicity of infection (MOI) of 3 for 72 h or 96 h and measured the amounts of luciferase produced in cell lysates
Summary
Cellular restriction factors play an important role in limiting the growth of a virus in a certain cell type. SAMHD1 has been shown to restrict the growth of other RNA or DNA viruses, such as human T cell leukemia virus type 1 (Sze et al, 2013), hepatitis B virus (Chen et al, 2014; Jeong et al, 2016; Sommer et al, 2016), vaccinia virus (Hollenbaugh et al, 2013), herpes simplex virus type-1 (Hollenbaugh et al, 2013; Kim et al, 2013; Badia et al, 2016), murine cytomegalovirus (MCMV) (Deutschmann et al, 2019), and human cytomegalovirus (HCMV) (Businger et al, 2019; Kim et al, 2019). The HCMV or MCMV-encoded kinases phosphorylate SAMHD1, counteracting the antiviral activity of SAMHD1 (Businger et al, 2019; Deutschmann et al, 2019; Kim et al, 2019)
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