A Hsp40 Chaperone Protein Interacts with and Modulates the Cellular Distribution of the Primase Protein of Human Cytomegalovirus

Abstract

Genomic DNA replication is a universal and essential process for all herpesvirus including human cytomegalovirus (HCMV). HCMV UL70 protein, which is believed to encode the primase activity of the viral DNA replication machinery and is highly conserved among herpesviruses, needs to be localized in the nucleus, the site of viral DNA synthesis. No host factors that facilitate the nuclear import of UL70 have been reported. In this study, we provided the first direct evidence that UL70 specifically interacts with a highly conserved and ubiquitously expressed member of the heat shock protein Hsp40/DNAJ family, DNAJB6, which is expressed as two isoforms, a and b, as a result of alternative splicing. The interaction of UL70 with a common region of DNAJB6a and b was identified by both a two hybrid screen in yeast and coimmunoprecipitation in human cells. In transfected cells, UL70 was primarily co-localized with DNAJB6a in the nuclei and with DNAJB6b in the cytoplasm, respectively. The nuclear import of UL70 was increased in cells in which DNAJB6a was up-regulated or DNAJB6b was down-regulated, and was reduced in cells in which DNAJB6a was down-regulated or DNAJB6b was up-regulated. Furthermore, the level of viral DNA synthesis and progeny production was increased in cells in which DNAJB6a was up-regulated or DNAJB6b was down-regulated, and was reduced in cells in which DNAJB6a was down-regulated or DNAJB6b was up-regulated. Thus, DNAJB6a and b appear to enhance the nuclear import and cytoplasmic accumulation of UL70, respectively. Our results also suggest that the relative expression levels of DNAJB6 isoforms may play a key role in regulating the cellular localization of UL70, leading to modulation of HCMV DNA synthesis and lytic infection.

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

“…Understanding the potential interactions between viral proteins and those between viral and human proteins is important for elucidating the mechanisms of infection and developing novel strategies for the treatment and prevention of herpesvirus latency and infection (14)(15)(16). 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).…”

Regulated Transport into the Nucleus of Herpesviridae DNA Replication Core Proteins

“…Indeed, ectopic expression of any of these three subunits in isolation has always been reported to be cytoplasmic [61,63,64,109], despite the fact that all three clearly need to be translocated to the nucleus in order to perform their role in viral replication, and can be readily detected in the nucleus of infected cells [109,110]. In all studies to date, the simultaneous expression of at least all three subunits appears to be necessary for nuclear targeting; in the case of HSV-1 and EBV, coexpression of the three subunits appears to be sufficient for nuclear targeting of the complexes [61,63,64].…”

“…In the case of KSHV, this scenario is completed by the evidence that PAF (ORF40/41) can also be partially translocated to the nucleus when expressed in the presence of other viral proteins, such as K8 or MTA, implying that piggy-back transport through individual viral factors may also contribute to nuclear accumulation [62]. The IMPs responsible have not been identified, but a recent report has implicated the cellular chaperone DNAJB6 in the nuclear transport of HCMV primase (UL70) [109]. Significantly, DNAB6 is not the only cellular chaperone playing a role in the nuclear targeting of herpesviral replicating proteins (see below).…”

“…While DNAJB6-a is nuclear, DNAJB6-b localizes exclusively in the cytoplasm, possibly through the fact that DNAJB6-b lacks the C -terminal domain, which contains a functional NLS [113]. Both isoforms interact with UL70, as demonstrated in the yeast two hybrid system and coimmunoprecipitations, suggesting that the interaction does not require the C -terminal domain of DNAJB6 [109]. The relative levels of DNAJB6 isoforms appear to play a crucial role in regulating the subcellular localization of HCMV UL70 protein.…”

Salmonella Virulence Factor SsrAB Regulated Factor Modulates Inflammatory Responses by Enhancing the Activation of NF-κB Signaling Pathway

“…Bands were detected with ECL reagent and quantitated using a Gel Documentation Station (Bio-Rad, Hercules, CA). The mouse anti-SrfA mAb was self-prepared by modifying a previously described procedure (41).…”