0508898 - ÚOCHB 2020 RIV US eng J - Článek v odborném periodiku
Ramakrishnan, D. - Xing, W. - Beran, R. K. - Chemuru, S. - Rohrs, H. - Niedziela-Majka, A. - Marchand, B. - Mehra, U. - Zábranský, Aleš - Doležal, Michal - Hubálek, Martin - Pichová, Iva - Gross, M. L. - Kwon, H. J. - Fletcher, S. P.
Hepatitis B Virus X Protein Function Requires Zinc Binding.
Journal of Virology. Roč. 93, č. 16 (2019), č. článku e00250-19. ISSN 0022-538X. E-ISSN 1098-5514
Grant CEP: GA MŠMT(CZ) EF16_019/0000729
Institucionální podpora: RVO:61388963
Klíčová slova: DDB1 * HBx * hepatitis B virus * Smc5/6 complex
Obor OECD: Virology
Impakt faktor: 4.501, rok: 2019
Způsob publikování: Open access
https://jvi.asm.org/content/93/16/e00250-19

The host structural maintenance of chromosomes 5/6 complex (Smc5/6) suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the cellular DNA damage-binding protein 1 (DDB1)-containing E3 ubiquitin ligase to target Smc5/6 for degradation. However, the details of how HBx modulates the interaction between DDB1 and Smc5/6 remain to be determined. In this study, we performed biophysical analyses of recombinant HBx and functional analysis of HBx mutants in HBV-infected primary human hepatocytes (PHH) to identify key regions and residues that are required for HBx function. We determined that recombinant HBx is soluble and exhibits stoichiometric zinc binding when expressed in the presence of DDB1. Mass spectrometry-based hydrogen-deuterium exchange and cysteine-specific chemical footprinting of the HBx: DDB1 complex identified several HBx cysteine residues (located between amino acids 61 and 137) that are likely involved in zinc binding. These cysteine residues did not form disulfide bonds in HBx expressed in human cells. In line with the biophysical data, functional analysis demonstrated that HBx amino acids 45 to 140 are required for Smc6 degradation and HBV transcription in PHH. Furthermore, site-directed mutagenesis determined that C61, C69, C137, and H139 are necessary for HBx function, although they are likely not essential for DDB1 binding. This CCCH motif is highly conserved in HBV as well as in the X proteins from various mammalian hepadnaviruses. Collectively, our data indicate that the essential HBx cysteine and histidine residues form a zinc-binding motif that is required for HBx function.
Trvalý link: http://hdl.handle.net/11104/0299694