Human Adenovirus Infection Causes Cellular E3 Ubiquitin Ligase MKRN1 Degradation Involving the Viral Core Protein pVII

Abstract

Human adenoviruses (HAdVs) are common human pathogens encoding a highly abundant histone-like core protein, VII, which is involved in nuclear delivery and protection of viral DNA as well as in sequestering immune danger signals in infected cells. The molecular details of how protein VII acts as a multifunctional protein have remained to a large extent enigmatic. Here we report the identification of several cellular proteins interacting with the precursor pVII protein. We show that the cellular E3 ubiquitin ligase MKRN1 is a novel precursor pVII-interacting protein in HAdV-C5-infected cells. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the late phase of HAdV-C5 infection in various human cell lines. MKRN1 protein degradation occurred independently of the HAdV E1B55K and E4orf6 proteins. We provide experimental evidence that the precursor pVII protein binding enhances MKRN1 self-ubiquitination, whereas the processed mature VII protein is deficient in this function. Based on these data, we propose that the pVII protein binding promotes MKRN1 self-ubiquitination, followed by proteasomal degradation of the MKRN1 protein, in HAdV-C5-infected cells. In addition, we show that measles virus and vesicular stomatitis virus infections reduce the MKRN1 protein accumulation in the recipient cells. Taken together, our results expand the functional repertoire of the HAdV-C5 precursor pVII protein in lytic virus infection and highlight MKRN1 as a potential common target during different virus infections. Human adenoviruses (HAdVs) are common pathogens causing a wide range of diseases. To achieve pathogenicity, HAdVs have to counteract a variety of host cell antiviral defense systems, which would otherwise hamper virus replication. In this study, we show that the HAdV-C5 histone-like core protein pVII binds to and promotes self-ubiquitination of a cellular E3 ubiquitin ligase named MKRN1. This mutual interaction between the pVII and MKRN1 proteins may prime MKRN1 for proteasomal degradation, because the MKRN1 protein is efficiently degraded during the late phase of HAdV-C5 infection. Since MKRN1 protein accumulation is also reduced in measles virus- and vesicular stomatitis virus-infected cells, our results signify the general strategy of viruses to target MKRN1.

Cellular Zinc Finger Protein 622 Hinders Human Adenovirus Lytic Growth and Limits Binding of the Viral pVII Protein to Virus DNA

“…Plasmids. Human full-length ZNF622 (NM_033414.2) cDNA (GenScript) was cloned into pcDNA3.1-HA plasmid (46) to express the C-terminal HA-tagged ZNF622 protein. ZNF622 mutants (1-108, 109-477, and 1-359) were generated with the PCR-mediated DNA deletion approach and cloned into the pcDNA3.1-HA background vector.…”

“…High-throughput protein-protein interaction experiments have revealed multiple cellular proteins interacting with the pVII protein in noninfected cells (40,46). However, only a few of these protein-protein interactions have been functionally characterized in HAdV-infected cells.…”

“…However, only a few of these protein-protein interactions have been functionally characterized in HAdV-infected cells. Since we have previously identified ZNF622 as a potential pVII interacting protein in a yeast two-hybrid screen (46), we aimed to disclose the functional impact of this interaction in HAdV-C5 infection. fection was analyzed by Western blotting.…”

CRISPR/Cas9-based inactivation of human papillomavirus oncogenes E6 and E7 induces senescence in cervical cancer cells

“…As evident from the quantification, the E6 and E7 knockout HeLa cells exhibited reduced lamin B1 levels and increased γ-H2AX nuclear foci signal relative to control treated HeLa cells. Loss of lamin B1 would compromise the integrity of nucleus, which leads to detection of cytoplasmic chromatin fragments (19,20). To test this, we checked for presence of cytoplasmic chromatin fragments by DAPI stained fragments in the cytoplasm (Fig.…”

“…The cells were lysed in buffer containing 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1 % SDS, supplemented with protease inhibitor (Complete Mini EDTA-free, Roche Applied Sciences). The lysates were incubated on ice for 20 min, sonicated and cleared by centrifugation at maximum speed for 10 min, 4°C (19). The supernatants were quantified using Bradford assay and subjected to electrophoresis using 8%-15% homemade gradient gels.…”