Modulation of the Cellular Distribution of Human Cytomegalovirus Helicase by Cellular Factor Snapin

Abstract

bControlled regulation of genomic DNA synthesis is a universally conserved process for all herpesviruses, including human cytomegalovirus (HCMV), and plays a key role in viral pathogenesis, such as persistent infections. HCMV UL105 is believed to encode the helicase of the DNA replication machinery that needs to localize in the nuclei, the site of viral DNA synthesis. No host factors that interact with UL105 have been identified. In this study, we show that UL105 specifically interacts with Snapin, a human protein that is predominantly localized in the cytoplasm and associated with cellular vesicles. UL105 was found to interact with Snapin in both the yeast two-hybrid screen and coimmunoprecipitation experiments in HCMV-infected cells. The nuclear and cytoplasmic levels of UL105 were decreased and increased in cells overexpressing Snapin, respectively, while the levels of UL105 in the nuclei and cytoplasm were increased and decreased in cells in which the expression of Snapin was downregulated with anti-Snapin small interfering RNA (siRNA) molecules, respectively. Furthermore, viral DNA synthesis and progeny production were decreased in cells overexpressing Snapin and increased in the anti-Snapin siRNA-treated cells, respectively. Our results provide the first direct evidence to suggest that Snapin interacts with UL105 and alters its cellular distribution, leading to modulation of viral DNA synthesis and progeny production. Our study further suggests that modulation of the cellular distribution of viral helicase by Snapin may represent a possible mechanism for regulating HCMV genomic DNA synthesis, a key step during herpesvirus lytic and persistent infections.

Interaction between the human cytomegalovirus-encoded UL142 and cellular Snapin proteins

“…Zhou et al (30), have revealed that Snapin has a critical role in coordinating dynein-driven retrograde transport and late endosomal-lysosomal trafficking, thus maintaining efficient autophagy-lysosomal function. In addition, a number of studies have demonstrated that Snapin may regulate HCMV genomic DNA synthesis by modulating the cellular distribution of viral helicase (25,(31)(32)(33).…”

“…As a novel HCMV encoded major histocompatibility complex (MHC) class I-related molecule, the UL142 protein contains an MHC class I Antigen (MICA) recognition domain (17,18), which is able to downregulate the expression levels of the NKG2D ligand, leading to protection from NK cytotoxicity and inhibition of NK cell-mediated lysis (19)(20)(21)(22). Snapin, expressed in a number of types of cells, including adipocytes and neuronal cells (23)(24)(25)(26), has been established to be associated with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex (27,28). SNAREs are ubiquitous proteins that direct vesicular trafficking and exocytosis, and mediate the release of neurotransmitters in neurons (29).…”

Human Cytomegalovirus Encoded miR-US25-1-5p Attenuates CD147/EMMPRIN-Mediated Early Antiviral Response

“…Human foreskin fibroblasts (HFF cells, passages 9 to 14), Human astrocytoma U251 cells (U251 cells), and Human Embryonic Kidney 293 cells (293 cells) were maintained in Dulbecco’s modified Eagle medium (DMEM), containing 10% fetal bovine serum, as previously described [ 19 , 20 ]. Herpes simplex virus 1 (HSV-1) was provided by Fenyong Liu (University of California, Berkeley, CA, USA).…”

“…Viral DNA was extracted from mock or HCMV-infected cells using a DNeasy tissue kit (Qiagen, Hilden, Germany). Intracellular viral DNAs were quantified using primers and a probe for amplifying the HCMV UL83 DNA region, as previously described [ 19 ]. The reaction was performed in a 20-μL amplification mixture (2 μL of DNA extract, 10 μL of 2 × Premix Ex Taq TM (TaKaRa, Dalian, China), 0.4 μL each of primer at 10 μM, 0.8 μL of the fluorogenic probe at 5 μM, 0.4 μL 50 × ROX Reference Dye II) using an ABI 7500 device (Applied Biosystems Inc., Foster City, CA, USA).…”

“…The titers of the viral stocks were determined in 1 × 10 5 HFF cells, with standard virus plaque assay and the number of plaques were counted 10 to 14 days after viral infection. The values obtained were averages ± SD from triplicate experiments [ 19 ].…”