Abstract:
: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.IMPORTANCE The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses HBV transcription. HBV counters this restriction by expressing HBV X protein (HBx), which redirects a host ubiquitin ligase to target Smc5/6 for degradation. Despite this recent advance in understanding HBx function, the key regions and residues of HBx required for Smc5/6 degradation have not been determined. In the present study, we performed biochemical, biophysical, and cell-based analyses of HBx. By doing so, we mapped the minimal functional region of HBx and identified a highly conserved CCCH motif in HBx that is likely responsible for coordinating zinc and is essential for HBx function. We also developed a method to produce soluble recombinant HBx protein that likely adopts a physiologically relevant conformation. Collectively, this study provides new insights into the HBx structure-function relationship and suggests a new approach for structural studies of this enigmatic viral regulatory protein.
journal_name
J Viroljournal_title
Journal of virologyauthors
Ramakrishnan D,Xing W,Beran RK,Chemuru S,Rohrs H,Niedziela-Majka A,Marchand B,Mehra U,Zábranský A,Doležal M,Hubálek M,Pichová I,Gross ML,Kwon HJ,Fletcher SPdoi
10.1128/JVI.00250-19subject
Has Abstractpub_date
2019-07-30 00:00:00issue
16eissn
0022-538Xissn
1098-5514pii
JVI.00250-19journal_volume
93pub_type
杂志文章abstract::Fusion of the membrane of the Moloney murine leukemia virus (Mo-MLV) Env protein is facilitated by cleavage of the R peptide from the cytoplasmic tail of its TM subunit, but the mechanism for this effect has remained obscure. The fusion is also controlled by the isomerization of the intersubunit disulfide of the Env S...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.02039-07
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.01178-06
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doi:10.1128/JVI.01790-10
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.20.2.465-477.1976
更新日期:1976-11-01 00:00:00
abstract::When Epstein-Barr virus (EBV) negative cells (Raji) were treated with iododeoxyuridine, only the early antigen (EA) component was induced. There was no significant increase in EBV DNA and no virus particles were observed. Somatic-cell hybrids were prepared from the fusion of Raji and D98 cells (D98/Raji). When these c...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.14.1.174-176.1974
更新日期:1974-07-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.80.7.3684-3691.2006
更新日期:2006-04-01 00:00:00
abstract::A pediatric human immunodeficiency virus type 1 (HIV-1) vaccine would be desirable to protect infants against HIV-1 transmission from breast-feeding. Such a vaccine would need to induce protective immunity at mucosal surfaces in neonates as soon as possible after birth. Recombinant adenovirus (rAd) vectors have been s...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.00512-12
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abstract::The human cytidine deaminase Apobec3F (h-A3F), a protein related to the previously recognized antiviral factor Apobec3G (h-A3G), has antiviral activity against human immunodeficiency virus type 1 (HIV-1) that is suppressed by the viral protein Vif. The mechanism of HIV-1 Vif-mediated suppression of h-A3F is not fully ...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.79.15.9579-9587.2005
更新日期:2005-08-01 00:00:00
abstract::A poliovirus-specific polyuridylic acid [poly(U)] polymerase that copies a polyadenylic acid template complexed to an oligouridylic acid primer was isolated from the membrane fraction of infected HeLa cells and was found to sediment at 4 to 5S on a linear 5 to 20% glycerol gradient. When the poly(U) polymerase was iso...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.29.1.352-360.1979
更新日期:1979-01-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/jvi.77.23.12679-12691.2003
更新日期:2003-12-01 00:00:00
abstract::Recruitment and extravasation of T cells through the blood-brain barrier are favored by adhesion molecule-mediated interactions of circulating T cells with endothelial cells. Since a common pathological finding in human T-cell leukemia virus type 1 (HTLV-1)-associated diseases is the infiltration of HTLV-1-infected T ...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.71.11.8522-8530.1997
更新日期:1997-11-01 00:00:00
abstract::The mechanism by which herpes simplex virus 1 (HSV-1) establishes latency in sensory neurons is largely unknown. Recent studies indicate that epigenetic modifications of the chromatin associated with the latent genome may play a key role in the transcriptional control of lytic genes during latency. In this study, we f...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.00686-09
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abstract::We set out to test the hypothesis that interleukin-22 (IL-22), a cytokine crucial for epithelial cell homeostasis and recovery from tissue injury, would be protective during influenza virus infection. Recent studies have identified phenotypically and functionally unique intestinal NK cells capable of producing the cyt...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.00187-10
更新日期:2010-08-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.66.11.6541-6546.1992
更新日期:1992-11-01 00:00:00
abstract::Natural killer T (NKT) cells are unique T lymphocytes that recognize CD1d-bound lipid antigens and play an important role in both innate and acquired immune responses against infectious diseases and tumors. We have already shown that a vesicular stomatitis virus (VSV) infection results in the rapid inhibition of murin...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.00881-08
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abstract::In this study, we show that replication-competent subgenomic hepatitis C virus (HCV) RNA can be transferred to permissive Huh7 cells, leading to the establishment of viral RNA replication. Further, we show that these events are mediated by exosomes rather than infectious virus particles. If similar events occur in viv...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.02721-14
更新日期:2015-03-01 00:00:00
abstract::Epstein-Barr virus (EBV) in vivo is known to establish persistent infection in resting, circulating memory B cells and to productively replicate in plasma cells. Until now, the molecular mechanism of how EBV switches from latency to lytic replication in vivo was not known. Here, we report that the plasma cell differen...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.01055-07
更新日期:2007-12-01 00:00:00
abstract::In some retroviruses, such as Rous sarcoma virus and prototype foamy virus, Gag proteins are known to shuttle between the nucleus and the cytoplasm and are implicated in nuclear export of the viral genomic unspliced RNA (gRNA) for subsequent encapsidation. A similar function has been proposed for human immunodeficienc...
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pub_type: 杂志文章
doi:10.1128/JVI.58.2.468-474.1986
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.67.6.3134-3141.1993
更新日期:1993-06-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.69.3.1480-1484.1995
更新日期:1995-03-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/jvi.74.12.5629-5638.2000
更新日期:2000-06-01 00:00:00
abstract::The feline homolog of the alpha-chemokine receptor CXCR4 has recently been shown to support cell-cell fusion mediated by CXCR4-dependent strains of human immunodeficiency virus (HIV) and strains of feline immunodeficiency virus (FIV) that have been selected for growth in the Crandell feline kidney (CrFK) cell line. In...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.72.8.6475-6481.1998
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abstract::The biologically relevant form of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein is oligomeric, with the major points of contact between oligomeric partners located in the ectodomain of gp41. To identify and map conserved epitopes and regions in gp41 where structure is influenced by quater...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.71.4.2674-2684.1997
更新日期:1997-04-01 00:00:00
abstract::Human endogenous retrovirus K (HERV-K) is the most intact retrovirus in the human genome. However, no single HERV-K provirus in the human genome today appears to be infectious. Since the Gag protein is the central component for the production of retrovirus particles, we investigated the abilities of Gag from two HERV-...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.01439-08
更新日期:2009-01-01 00:00:00
abstract::The phenotypic properties of ICP27 temperature-sensitive and deletion mutants and the results of transient expression assays have demonstrated that ICP27 has a modulatory effect on viral gene expression induced by ICPs 0 and 4. In order to identify the regions of the ICP27 molecule that are responsible for its enhanci...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.64.7.3471-3485.1990
更新日期:1990-07-01 00:00:00
abstract::During lytic infection, the genome of herpes simplex virus 1 (HSV-1) is associated with limited levels of histones but does not form a regular repeating nucleosomal structure. However, the previous observation that chromatin remodeling factors are recruited into viral replication compartments indicates that chromatin ...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.01575-08
更新日期:2008-12-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.75.23.11583-11593.2001
更新日期:2001-12-01 00:00:00
abstract::The murine monoclonal antibody (MAb) 5A8, which is reactive with domain 2 of CD4, blocks human immunodeficiency virus type 1 (HIV-1) infection and syncytium formation of CD4+ cells (L. C. Burkly, D. Olson, R. Shapiro, G. Winkler, J. J. Rosa, D. W. Thomas, C. Williams, and P. Chisholm, J. Immunol., in press). Here we s...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.66.8.4784-4793.1992
更新日期:1992-08-01 00:00:00
abstract::Human cytomegalovirus (HCMV) is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and newborn infants infected in utero. The viral envelope glycoprotein B (gB) is an attractive molecule for active vaccination and passive immunoprophylaxis and therapy. Using human monoclonal an...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.00434-13
更新日期:2013-08-01 00:00:00