Publications by authors named "Hongjie Xia"

36 Publications

A trans-complementation system for SARS-CoV-2 recapitulates authentic viral replication without virulence.

Cell 2021 Feb 23. Epub 2021 Feb 23.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX, USA; Sealy Institute for Vaccine Sciences, University of Texas Medical Branch, Galveston, TX, USA; Sealy Center for Structural Biology & Molecular Biophysics, University of Texas Medical Branch, Galveston, TX, USA. Electronic address:

The biosafety level 3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research. Here, we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing ORF3 and envelope gene deletions, as well as mutated transcriptional regulator sequences, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. Thus, the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.
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http://dx.doi.org/10.1016/j.cell.2021.02.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901297PMC
February 2021

Inhibition of innate immune response ameliorates Zika virus-induced neurogenesis deficit in human neural stem cells.

PLoS Negl Trop Dis 2021 Mar 3;15(3):e0009183. Epub 2021 Mar 3.

Department of Neuroscience, Cell Biology and Anatomy, University of Texas Medical Branch, Galveston, Texas, United States of America.

Global Zika virus (ZIKV) outbreaks and their strong link to microcephaly have raised major public health concerns. ZIKV has been reported to affect the innate immune responses in neural stem/progenitor cells (NS/PCs). However, it is unclear how these immune factors affect neurogenesis. In this study, we used Asian-American lineage ZIKV strain PRVABC59 to infect primary human NS/PCs originally derived from fetal brains. We found that ZIKV overactivated key molecules in the innate immune pathways to impair neurogenesis in a cell stage-dependent manner. Inhibiting the overactivated innate immune responses ameliorated ZIKV-induced neurogenesis reduction. This study thus suggests that orchestrating the host innate immune responses in NS/PCs after ZIKV infection could be promising therapeutic approach to attenuate ZIKV-associated neuropathology.
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http://dx.doi.org/10.1371/journal.pntd.0009183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7959377PMC
March 2021

Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and N501Y variants by BNT162b2 vaccine-elicited sera.

Nat Med 2021 Feb 8. Epub 2021 Feb 8.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA.

We engineered three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses containing key spike mutations from the newly emerged United Kingdom (UK) and South African (SA) variants: N501Y from UK and SA; 69/70-deletion + N501Y + D614G from UK; and E484K + N501Y + D614G from SA. Neutralization geometric mean titers (GMTs) of 20 BTN162b2 vaccine-elicited human sera against the three mutant viruses were 0.81- to 1.46-fold of the GMTs against parental virus, indicating small effects of these mutations on neutralization by sera elicited by two BNT162b2 doses.
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http://dx.doi.org/10.1038/s41591-021-01270-4DOI Listing
February 2021

Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K, and N501Y variants by BNT162b2 vaccine-elicited sera.

bioRxiv 2021 Jan 27. Epub 2021 Jan 27.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston TX, U.S.A.

We engineered three SARS-CoV-2 viruses containing key spike mutations from the newly emerged United Kingdom (UK) and South African (SA) variants: N501Y from UK and SA; 69/70-deletion+N501Y+D614G from UK; and E484K+N501Y+D614G from SA. Neutralization geometric mean titers (GMTs) of twenty BTN162b2 vaccine-elicited human sera against the three mutant viruses were 0.81- to 1.46-fold of the GMTs against parental virus, indicating small effects of these mutations on neutralization by sera elicited by two BNT162b2 doses.
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http://dx.doi.org/10.1101/2021.01.27.427998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852264PMC
January 2021

A -complementation system for SARS-CoV-2.

bioRxiv 2021 Jan 19. Epub 2021 Jan 19.

The biosafety level-3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research and countermeasure development. Here we report a -complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The -complementation system consists of two components: a genomic viral RNA containing a deletion of ORF3 and envelope gene, and a producer cell line expressing the two deleted genes. complementation of the two components generates virions that can infect naive cells for only one round, but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. The results suggest that the -complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.
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http://dx.doi.org/10.1101/2021.01.16.426970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836106PMC
January 2021

Ubiquitination of SARS-CoV-2 ORF7a promotes antagonism of interferon response.

Cell Mol Immunol 2021 03 20;18(3):746-748. Epub 2021 Jan 20.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, 77555, USA.

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http://dx.doi.org/10.1038/s41423-020-00603-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815971PMC
March 2021

Neutralization of N501Y mutant SARS-CoV-2 by BNT162b2 vaccine-elicited sera.

Res Sq 2021 Jan 13. Epub 2021 Jan 13.

Rapidly spreading variants of SARS-CoV-2 that have arisen in the United Kingdom and South Africa share the spike N501Y substitution, which is of particular concern because it is located in the viral receptor binding site for cell entry and increases binding to the receptor. We generated isogenic N501 and Y501 SARS-CoV-2. Twenty human sera from the mRNA-based vaccine BNT162b2 trial exhibited equivalent neutralizing titers to the N501 and Y501 viruses.
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http://dx.doi.org/10.21203/rs.3.rs-143532/v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814835PMC
January 2021

Neutralization of N501Y mutant SARS-CoV-2 by BNT162b2 vaccine-elicited sera.

bioRxiv 2021 Jan 7. Epub 2021 Jan 7.

Rapidly spreading variants of SARS-CoV-2 that have arisen in the United Kingdom and South Africa share the spike N501Y substitution, which is of particular concern because it is located in the viral receptor binding site for cell entry and increases binding to the receptor (angiotensin converting enzyme 2). We generated isogenic N501 and Y501 SARS-CoV-2. Sera of 20 participants in a previously reported trial of the mRNA-based COVID-19 vaccine BNT162b2 had equivalent neutralizing titers to the N501 and Y501 viruses.
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http://dx.doi.org/10.1101/2021.01.07.425740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7805448PMC
January 2021

Antagonism of Type I Interferon by Severe Acute Respiratory Syndrome Coronavirus 2.

J Interferon Cytokine Res 2020 12;40(12):543-548

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA.

The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), warranting urgent study of the molecular mechanisms of SARS-CoV-2 infection and host immune response. Type I interferon (IFN-I) is a key component of host innate immune system responsible for eliminating the virus at the early stage of infection. In contrast, SARS-CoV-2 has evolved multiple strategies to evade innate immune response to facilitate viral replication, transmission, and pathogenesis. This review summarizes the recent progresses on SARS-CoV-2 proteins that antagonize host IFN-I production and/or signaling. These progresses have provided knowledge for new vaccine and antiviral development to prevent and control COVID-19.
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http://dx.doi.org/10.1089/jir.2020.0214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757701PMC
December 2020

Spike mutation D614G alters SARS-CoV-2 fitness.

Nature 2021 04 26;592(7852):116-121. Epub 2020 Oct 26.

Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX, USA.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein substitution D614G became dominant during the coronavirus disease 2019 (COVID-19) pandemic. However, the effect of this variant on viral spread and vaccine efficacy remains to be defined. Here we engineered the spike D614G substitution in the USA-WA1/2020 SARS-CoV-2 strain, and found that it enhances viral replication in human lung epithelial cells and primary human airway tissues by increasing the infectivity and stability of virions. Hamsters infected with SARS-CoV-2 expressing spike(D614G) (G614 virus) produced higher infectious titres in nasal washes and the trachea, but not in the lungs, supporting clinical evidence showing that the mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increase transmission. Sera from hamsters infected with D614 virus exhibit modestly higher neutralization titres against G614 virus than against D614 virus, suggesting that the mutation is unlikely to reduce the ability of vaccines in clinical trials to protect against COVID-19, and that therapeutic antibodies should be tested against the circulating G614 virus. Together with clinical findings, our work underscores the importance of this variant in viral spread and its implications for vaccine efficacy and antibody therapy.
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http://dx.doi.org/10.1038/s41586-020-2895-3DOI Listing
April 2021

Evasion of Type I Interferon by SARS-CoV-2.

Cell Rep 2020 10 19;33(1):108234. Epub 2020 Sep 19.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555, USA; Sealy Institute for Vaccine Sciences, University of Texas Medical Branch, Galveston, TX 77555, USA; Sealy Center for Structural Biology & Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA. Electronic address:

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication and host immune response determine coronavirus disease 2019 (COVID-19), but studies evaluating viral evasion of immune response are lacking. Here, we use unbiased screening to identify SARS-CoV-2 proteins that antagonize type I interferon (IFN-I) response. We found three proteins that antagonize IFN-I production via distinct mechanisms: nonstructural protein 6 (nsp6) binds TANK binding kinase 1 (TBK1) to suppress interferon regulatory factor 3 (IRF3) phosphorylation, nsp13 binds and blocks TBK1 phosphorylation, and open reading frame 6 (ORF6) binds importin Karyopherin α 2 (KPNA2) to inhibit IRF3 nuclear translocation. We identify two sets of viral proteins that antagonize IFN-I signaling through blocking signal transducer and activator of transcription 1 (STAT1)/STAT2 phosphorylation or nuclear translocation. Remarkably, SARS-CoV-2 nsp1 and nsp6 suppress IFN-I signaling more efficiently than SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Thus, when treated with IFN-I, a SARS-CoV-2 replicon replicates to a higher level than chimeric replicons containing nsp1 or nsp6 from SARS-CoV or MERS-CoV. Altogether, the study provides insights on SARS-CoV-2 evasion of IFN-I response and its potential impact on viral transmission and pathogenesis.
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http://dx.doi.org/10.1016/j.celrep.2020.108234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501843PMC
October 2020

Spike mutation D614G alters SARS-CoV-2 fitness and neutralization susceptibility.

Res Sq 2020 Sep 10. Epub 2020 Sep 10.

The University of Texas Medical Branch at Galveston.

A spike protein mutation D614G became dominant in SARS-CoV-2 during the COVID-19 pandemic. However, the mutational impact on viral spread and vaccine efficacy remains to be defined. Here we engineer the D614G mutation in the SARS-CoV-2 USA-WA1/2020 strain and characterize its effect on viral replication, pathogenesis, and antibody neutralization. The D614G mutation significantly enhances SARS-CoV-2 replication on human lung epithelial cells and primary human airway tissues, through an improved infectivity of virions with the spike receptor-binding domain in an "up" conformation for binding to ACE2 receptor. Hamsters infected with D614 or G614 variants developed similar levels of weight loss. However, the G614 virus produced higher infectious titers in the nasal washes and trachea, but not lungs, than the D614 virus. The hamster results confirm clinical evidence that the D614G mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increases transmission. For antibody neutralization, sera from D614 virus-infected hamsters consistently exhibit higher neutralization titers against G614 virus than those against D614 virus, indicating that (i) the mutation may not reduce the ability of vaccines in clinical trials to protect against COVID-19 and (ii) therapeutic antibodies should be tested against the circulating G614 virus before clinical development.
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http://dx.doi.org/10.21203/rs.3.rs-70482/v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7491579PMC
September 2020

Association of leisure sedentary time with common chronic disease risk factors: A longitudinal study of China Health and Nutrition Surveys.

Int J Health Plann Manage 2021 Jan 9;36(1):100-112. Epub 2020 Sep 9.

Department of Medical Insurance Management, Renmin Hospital of Wuhan University, Wuhan, China.

Background: Although the common risk factors were identified and controlled for many years, the overall prevalence of chronic diseases continued to increase in China.

Objective: We presumed the leisure sedentariness as a latent but pivotal factor of chronic diseases, and examined its distribution and changing trend, analysed its interaction effects on common risk factors, which could provide a new perspective for the prevention and management.

Methods: A total of 5013 participants were screened out from China Health and Nutrition Survey. Random-effects ordered logistic models were used for ordinal dependent variables, and fixed-effects or random-effects logit models were used for binary dependent variables.

Results: From 2004 to 2011, the prevalence of high leisure sedentary time (LSED) increased by 58.58%. Members of the high LSED group were likely to choose fast food, salty snacks, soft drinks and more likely to smoke or drink alcohol compared with those of the low LSED group. However, they preferred walking, sports and body building more than those of the low LSED group.

Conclusions: For the unhealthy dietary, tobacco and alcohol consumption, more targeted introduction and guidance related to sedentary time should be promoted. Meanwhile, the appeal for physical exercise as well as adequate facilities should be initiated.
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http://dx.doi.org/10.1002/hpm.3070DOI Listing
January 2021

Spike mutation D614G alters SARS-CoV-2 fitness and neutralization susceptibility.

bioRxiv 2020 Sep 2. Epub 2020 Sep 2.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston TX, USA.

A spike protein mutation D614G became dominant in SARS-CoV-2 during the COVID-19 pandemic. However, the mutational impact on viral spread and vaccine efficacy remains to be defined. Here we engineer the D614G mutation in the SARS-CoV-2 USA-WA1/2020 strain and characterize its effect on viral replication, pathogenesis, and antibody neutralization. The D614G mutation significantly enhances SARS-CoV-2 replication on human lung epithelial cells and primary human airway tissues, through an improved infectivity of virions with the spike receptor-binding domain in an "up" conformation for binding to ACE2 receptor. Hamsters infected with D614 or G614 variants developed similar levels of weight loss. However, the G614 virus produced higher infectious titers in the nasal washes and trachea, but not lungs, than the D614 virus. The hamster results confirm clinical evidence that the D614G mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increases transmission. For antibody neutralization, sera from D614 virus-infected hamsters consistently exhibit higher neutralization titers against G614 virus than those against D614 virus, indicating that (i) the mutation may not reduce the ability of vaccines in clinical trials to protect against COVID-19 and (ii) therapeutic antibodies should be tested against the circulating G614 virus before clinical development.

Importance: Understanding the evolution of SARS-CoV-2 during the COVID-19 pandemic is essential for disease control and prevention. A spike protein mutation D614G emerged and became dominant soon after the pandemic started. By engineering the D614G mutation into an authentic wild-type SARS-CoV-2 strain, we demonstrate the importance of this mutation to (i) enhanced viral replication on human lung epithelial cells and primary human airway tissues, (ii) improved viral fitness in the upper airway of infected hamsters, and (iii) increased susceptibility to neutralization. Together with clinical findings, our work underscores the importance of this mutation in viral spread, vaccine efficacy, and antibody therapy.
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http://dx.doi.org/10.1101/2020.09.01.278689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480025PMC
September 2020

A Zika virus envelope mutation preceding the 2015 epidemic enhances virulence and fitness for transmission.

Proc Natl Acad Sci U S A 2020 08 3;117(33):20190-20197. Epub 2020 Aug 3.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555;

Arboviruses maintain high mutation rates due to lack of proofreading ability of their viral polymerases, in some cases facilitating adaptive evolution and emergence. Here we show that, just before its 2013 spread to the Americas, Zika virus (ZIKV) underwent an envelope protein V473M substitution (E-V473M) that increased neurovirulence, maternal-to-fetal transmission, and viremia to facilitate urban transmission. A preepidemic Asian ZIKV strain (FSS13025 isolated in Cambodia in 2010) engineered with the V473M substitution significantly increased neurovirulence in neonatal mice and produced higher viral loads in the placenta and fetal heads in pregnant mice. Conversely, an epidemic ZIKV strain (PRVABC59 isolated in Puerto Rico in 2015) engineered with the inverse M473V substitution reversed the pathogenic phenotypes. Although E-V473M did not affect oral infection of mosquitoes, competition experiments in cynomolgus macaques showed that this mutation increased its fitness for viremia generation, suggesting adaptive evolution for human viremia and hence transmission. Mechanistically, the V473M mutation, located at the second transmembrane helix of the E protein, enhances virion morphogenesis. Overall, our study revealed E-V473M as a critical determinant for enhanced ZIKV virulence, intrauterine transmission during pregnancy, and viremia to facilitate urban transmission.
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http://dx.doi.org/10.1073/pnas.2005722117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443865PMC
August 2020

A cocrystal structure of dengue capsid protein in complex of inhibitor.

Proc Natl Acad Sci U S A 2020 07 15;117(30):17992-18001. Epub 2020 Jul 15.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555;

Dengue virus (DENV) was designated as a top 10 public health threat by the World Health Organization in 2019. No clinically approved anti-DENV drug is currently available. Here we report the high-resolution cocrystal structure (1.5 Å) of the DENV-2 capsid protein in complex with an inhibitor that potently suppresses DENV-2 but not other DENV serotypes. The inhibitor induces a "kissing" interaction between two capsid dimers. The inhibitor-bound capsid tetramers are assembled inside virions, resulting in defective uncoating of nucleocapsid when infecting new cells. Resistant DENV-2 emerges through one mutation that abolishes hydrogen bonds in the capsid structure, leading to a loss of compound binding. Structure-based analysis has defined the amino acids responsible for the inhibitor's inefficacy against other DENV serotypes. The results have uncovered an antiviral mechanism through inhibitor-induced tetramerization of the viral capsid and provided essential structural and functional knowledge for rational design of panserotype DENV capsid inhibitors.
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http://dx.doi.org/10.1073/pnas.2003056117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395448PMC
July 2020

Envelope protein ubiquitination drives entry and pathogenesis of Zika virus.

Nature 2020 09 8;585(7825):414-419. Epub 2020 Jul 8.

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA.

Zika virus (ZIKV) belongs to the family Flaviviridae, and is related to other viruses that cause human diseases. Unlike other flaviviruses, ZIKV infection can cause congenital neurological disorders and replicates efficiently in reproductive tissues. Here we show that the envelope protein (E) of ZIKV is polyubiquitinated by the E3 ubiquitin ligase TRIM7 through Lys63 (K63)-linked polyubiquitination. Accordingly, ZIKV replicates less efficiently in the brain and reproductive tissues of Trim7 mice. Ubiquitinated E is present on infectious virions of ZIKV when they are released from specific cell types, and enhances virus attachment and entry into cells. Specifically, K63-linked polyubiquitin chains directly interact with the TIM1 (also known as HAVCR1) receptor of host cells, which enhances virus entry in cells as well as in brain tissue in vivo. Recombinant ZIKV mutants that lack ubiquitination are attenuated in human cells and in wild-type mice, but not in live mosquitoes. Monoclonal antibodies against K63-linked polyubiquitin specifically neutralize ZIKV and reduce viraemia in mice. Our results demonstrate that the ubiquitination of ZIKV E is an important determinant of virus entry, tropism and pathogenesis.
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http://dx.doi.org/10.1038/s41586-020-2457-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501154PMC
September 2020

Role of microglia in the dissemination of Zika virus from mother to fetal brain.

PLoS Negl Trop Dis 2020 07 6;14(7):e0008413. Epub 2020 Jul 6.

Department of Neuroscience, Cell Biology and Anatomy, University of Texas Medical Branch, Galveston, Texas, United States of America.

Global Zika virus (ZIKV) outbreaks and their link to microcephaly have raised major public health concerns. However, the mechanism of maternal-fetal transmission remains largely unknown. In this study, we determined the role of yolk sac (YS) microglial progenitors in a mouse model of ZIKV vertical transmission. We found that embryonic (E) days 6.5-E8.5 were a critical window for ZIKV infection that resulted in fetal demise and microcephaly, and YS microglial progenitors were susceptible to ZIKV infection. Ablation of YS microglial progenitors significantly reduced the viral load in both the YS and the embryonic brain. Taken together, these results support the hypothesis that YS microglial progenitors serve as "Trojan horses," contributing to ZIKV fetal brain dissemination and congenital brain defects.
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http://dx.doi.org/10.1371/journal.pntd.0008413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7365479PMC
July 2020

Zika Virus NS2A-Mediated Virion Assembly.

mBio 2019 10 29;10(5). Epub 2019 Oct 29.

Department of Biochemistry & Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA

The flavivirus virion consists of an envelope outer layer, formed by envelope (E) and membrane (M) proteins on a lipid bilayer, and an internal core, formed by capsid (C) protein and genomic RNA. The molecular mechanism of flavivirus assembly is not well understood. Here, we show that Zika virus (ZIKV) NS2A protein recruits genomic RNA, the structural protein prM/E complex, and the NS2B/NS3 protease complex to the virion assembly site and orchestrates virus morphogenesis. Coimmunoprecipitation analysis showed that ZIKV NS2A binds to prM, E, NS2B, and NS3 (but not C, NS4B, or NS5) in a viral RNA-independent manner, whereas prM/E complex does not interact with NS2B/NS3 complex. Remarkably, a single-amino-acid mutation (E103A) of NS2A impairs its binding to prM/E and NS2B/NS3 and abolishes virus production, demonstrating the indispensable role of NS2A/prM/E and NS2A/NS2B/NS3 interactions in virion assembly. In addition, RNA-protein pulldown analysis identified a stem-loop RNA from the 3' untranslated region (UTR) of the viral genome as an "RNA recruitment signal" for ZIKV assembly. The 3' UTR RNA binds to a cytoplasmic loop of NS2A protein. Mutations of two positively charged residues (R96A and R102A) from the cytoplasmic loop reduce NS2A binding to viral RNA, leading to a complete loss of virion assembly. Collectively, our results support a virion assembly model in which NS2A recruits viral NS2B/NS3 protease and structural C-prM-E polyprotein to the virion assembly site; once the C-prM-E polyprotein has been processed, NS2A presents viral RNA to the structural proteins for virion assembly. ZIKV is a recently emerged mosquito-borne flavivirus that can cause devastating congenital Zika syndrome in pregnant women and Guillain-Barré syndrome in adults. The molecular mechanism of ZIKV virion assembly is largely unknown. Here, we report that ZIKV NS2A plays a central role in recruiting viral RNA, structural protein prM/E, and viral NS2B/NS3 protease to the virion assembly site and orchestrating virion morphogenesis. One mutation that impairs these interactions does not significantly affect viral RNA replication but selectively abolishes virion assembly, demonstrating the specific role of these interactions in virus morphogenesis. We also show that the 3' UTR of ZIKV RNA may serve as a "recruitment signal" through binding to NS2A to enter the virion assembly site. Following a coordinated cleavage of C-prM-E at the virion assembly site, NS2A may present the viral RNA to C protein for nucleocapsid formation followed by envelopment with prM/E proteins. The results have provided new insights into flavivirus virion assembly.
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http://dx.doi.org/10.1128/mBio.02375-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6819661PMC
October 2019

Genetic and biochemical characterizations of Zika virus NS2A protein.

Emerg Microbes Infect 2019 ;8(1):585-602

c Department of Biochemistry & Molecular Biology , University of Texas Medical Branch , Galveston , TX , USA.

Zika virus (ZIKV) can cause devastating congenital Zika syndromes in pregnant women and Guillain-Barre syndrome in adults. Understanding the molecular mechanism of ZIKV replication is essential for antiviral and vaccine development. Here we report the structural and functional characterization of ZIKV NS2A protein. Biochemical structural probing suggests that ZIKV NS2A has a single segment that traverses the ER membrane and six segments that peripherally associate with the ER membrane. Functional analysis has defined distinct NS2A residues essential for viral RNA synthesis or virion assembly. Only the virion assembly-defective mutants, but not the RNA synthesis-defective mutants, could be rescued through trans complementation with a wide-type NS2A protein. These results suggest that the NS2A molecules in virion assembly complex could be recruited in trans, whereas the NS2A molecules in viral replication complex must be recruited in cis. Together with previous results, we propose a flavivirus assembly model where NS2A plays a central role in modulating viral structural and nonstructural proteins as well as genomic RNA during virion assembly.
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http://dx.doi.org/10.1080/22221751.2019.1598291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6455252PMC
June 2019

A Single-Dose Live-Attenuated Zika Virus Vaccine with Controlled Infection Rounds that Protects against Vertical Transmission.

Cell Host Microbe 2018 10;24(4):487-499.e5

Department of Biochemistry & Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA; Sealy Institute for Vaccine Sciences, University of Texas Medical Branch, Galveston, TX, USA; Sealy Center for Structural Biology & Molecular Biophysics, University of Texas Medical Branch, Galveston, TX, USA; Department of Pharmacology & Toxicology, University of Texas Medical Branch, Galveston, TX, USA. Electronic address:

Zika virus (ZIKV) infection of the mother during pregnancy causes devastating Zika congenital syndrome in the offspring. A ZIKV vaccine with optimal safety and immunogenicity for use in pregnant women is critically needed. Toward this goal, we have developed a single-dose live-attenuated vaccine candidate that infects cells with controlled, limited infection rounds. The vaccine contains a 9-amino-acid deletion in the viral capsid protein and replicates to titers of > 10 focus-forming units (FFU)/mL in cells expressing the full-length capsid protein. Immunization of A129 mice with one dose (10 FFU) did not produce viremia, but elicited protective immunity that completely prevented viremia, morbidity, and mortality after challenge with an epidemic ZIKV strain (10 PFU). A single-dose vaccination also fully prevented infection of pregnant mice and maternal-to-fetal transmission. Intracranial injection of the vaccine (10 FFU) to 1-day-old mice did not cause any disease or death, underscoring the safety of this vaccine candidate.
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http://dx.doi.org/10.1016/j.chom.2018.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6188708PMC
October 2018

An evolutionary NS1 mutation enhances Zika virus evasion of host interferon induction.

Nat Commun 2018 01 29;9(1):414. Epub 2018 Jan 29.

Department of Biochemistry & Molecular Biology, University of Texas Medical Branch, Galveston, TX, 77555, USA.

Virus-host interactions determine an infection outcome. The Asian lineage of Zika virus (ZIKV), responsible for the recent epidemics, has fixed a mutation in the NS1 gene after 2012 that enhances mosquito infection. Here we report that the same mutation confers NS1 to inhibit interferon-β induction. This mutation enables NS1 binding to TBK1 and reduces TBK1 phosphorylation. Engineering the mutation into a pre-epidemic ZIKV strain debilitates the virus for interferon-β induction; reversing the mutation in an epidemic ZIKV strain invigorates the virus for interferon-β induction; these mutational effects are lost in IRF3-knockout cells. Additionally, ZIKV NS2A, NS2B, NS4A, NS4B, and NS5 can also suppress interferon-β production through targeting distinct components of the RIG-I pathway; however, for these proteins, no antagonistic difference is observed among various ZIKV strains. Our results support the mechanism that ZIKV has accumulated mutation(s) that increases the ability to evade immune response and potentiates infection and epidemics.
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http://dx.doi.org/10.1038/s41467-017-02816-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5788864PMC
January 2018

Potential Mechanisms for Enhanced Zika Epidemic and Disease.

ACS Infect Dis 2018 05 22;4(5):656-659. Epub 2018 Jan 22.

A number of mechanisms have driven the explosive epidemics and severe diseases of Zika virus since 2007. Here, we comment on how herd immunity, heterologous flavivirus preimmunity, and viral mutations could enhance the epidemic potential and disease severity of Zika virus in humans.
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http://dx.doi.org/10.1021/acsinfecdis.8b00004DOI Listing
May 2018

RNA chaperones encoded by RNA viruses.

Virol Sin 2015 Dec 22;30(6):401-9. Epub 2015 Dec 22.

State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China.

RNAs are functionally diverse macromolecules whose proper functions rely strictly upon their correct tertiary structures. However, because of their high structural flexibility, correct folding of RNAs is challenging and slow. Therefore, cells and viruses encode a variety of RNA remodeling proteins, including helicases and RNA chaperones. In RNA viruses, these proteins are believed to play pivotal roles in all the processes involving viral RNAs during the life cycle. RNA helicases have been studied extensively for decades, whereas RNA chaperones, particularly virus-encoded RNA chaperones, are often overlooked. This review describes the activities of RNA chaperones encoded by RNA viruses, particularly the ones identified and characterized in recent years, and the functions of these proteins in different steps of viral life cycles, and presents an overview of this unique group of proteins.
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http://dx.doi.org/10.1007/s12250-015-3676-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7091039PMC
December 2015

Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone.

PLoS Pathog 2015 Jul 28;11(7):e1005067. Epub 2015 Jul 28.

State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China.

RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3'-to-5' unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our understanding of enteroviruses and the two types of RNA remodeling activities.
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http://dx.doi.org/10.1371/journal.ppat.1005067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4517893PMC
July 2015

The identification and characterization of nucleic acid chaperone activity of human enterovirus 71 nonstructural protein 3AB.

Virology 2014 Sep 9;464-465:353-364. Epub 2014 Aug 9.

State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, 430072 China. Electronic address:

Human enterovirus 71 (EV71) belongs to the genus Enterovirus in the family Picornaviridae and has been recognized as one of the most important pathogens that cause emerging infectious disease. Despite of the importance of EV71, the nonstructural protein 3AB from this virus is little understood for its function during EV71 replication. Here we expressed EV71 3AB protein as recombinant protein in a eukaryotic expression system and uncovered that this protein possesses a nucleic acid helix-destabilizing and strand annealing acceleration activity in a dose-dependent manner, indicating that EV71 3AB is a nucleic acid chaperone protein. Moreover, we characterized the RNA chaperone activity of EV71 3AB, and revealed that divalent metal ions, such as Mg(2+) and Zn(2+), were able to inhibit the RNA helix-destabilizing activity of 3AB to different extents. Moreover, we determined that 3B plus the last 7 amino acids at the C-terminal of 3A (termed 3B+7) possess the RNA chaperone activity, and five amino acids, i.e. Lys-80, Phe-82, Phe-85, Tyr-89, and Arg-103, are critical and probably the active sites of 3AB for its RNA chaperone activity. This report reveals that EV71 3AB displays an RNA chaperone activity, adds a new member to the growing list of virus-encoded RNA chaperones, and provides novel knowledge about the virology of EV71.
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http://dx.doi.org/10.1016/j.virol.2014.07.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7112070PMC
September 2014