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1.
PLoS Biol ; 22(2): e3002544, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38422166

RESUMEN

Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the cofactor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface, and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity. Treatment with the compounds reduced replication of infectious EBOV in cells and in vivo in a mouse model. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity.


Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Proteínas de la Nucleocápside , Ubiquitina , Replicación Viral , Animales , Humanos , Ratones , Ebolavirus/genética , Ubiquitina/metabolismo , Proteínas Reguladoras y Accesorias Virales , Replicación Viral/genética
2.
Nature ; 585(7825): 414-419, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32641828

RESUMEN

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 tissues1-3. 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.


Asunto(s)
Ubiquitinación , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Virus Zika/metabolismo , Virus Zika/patogenicidad , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Encéfalo/metabolismo , Línea Celular , Culicidae/citología , Culicidae/virología , Endosomas/metabolismo , Femenino , Receptor Celular 1 del Virus de la Hepatitis A/metabolismo , Humanos , Masculino , Fusión de Membrana , Ratones , Especificidad de Órganos , Poliubiquitina/inmunología , Poliubiquitina/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Tropismo Viral , Viremia/inmunología , Viremia/prevención & control , Viremia/virología , Replicación Viral , Virus Zika/química , Virus Zika/genética , Infección por el Virus Zika/prevención & control , Infección por el Virus Zika/virología
3.
PLoS Pathog ; 18(5): e1010532, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35533195

RESUMEN

Ebola virus (EBOV) VP35 is a polyfunctional protein involved in viral genome packaging, viral polymerase function, and host immune antagonism. The mechanisms regulating VP35's engagement in different functions are not well-understood. We previously showed that the host E3 ubiquitin ligase TRIM6 ubiquitinates VP35 at lysine 309 (K309) to facilitate virus replication. However, how K309 ubiquitination regulates the function of VP35 as the viral polymerase co-factor and the precise stage(s) of the EBOV replication cycle that require VP35 ubiquitination are not known. Here, we generated recombinant EBOVs encoding glycine (G) or arginine (R) mutations at VP35/K309 (rEBOV-VP35/K309G/-R) and show that both mutations prohibit VP35/K309 ubiquitination. The K309R mutant retains dsRNA binding and efficient type-I Interferon (IFN-I) antagonism due to the basic residue conservation. The rEBOV-VP35/K309G mutant loses the ability to efficiently antagonize the IFN-I response, while the rEBOV-VP35/K309R mutant's suppression is enhanced. The replication of both mutants was significantly attenuated in both IFN-competent and -deficient cells due to impaired interactions with the viral polymerase. The lack of ubiquitination on VP35/K309 or TRIM6 deficiency disrupts viral transcription with increasing severity along the transcriptional gradient. This disruption of the transcriptional gradient results in unbalanced viral protein production, including reduced synthesis of the viral transcription factor VP30. In addition, lack of ubiquitination on K309 results in enhanced interactions with the viral nucleoprotein and premature nucleocapsid packaging, leading to dysregulation of virus assembly. Overall, we identified a novel role of VP35 ubiquitination in coordinating viral transcription and assembly.


Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Ebolavirus/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Proteínas de la Nucleocápside/metabolismo , Ubiquitinación , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismo , Transcripción Viral
4.
Viruses ; 16(8)2024 Aug 12.
Artículo en Inglés | MEDLINE | ID: mdl-39205259

RESUMEN

The E3 ubiquitin ligase TRIM7 is known to have dual roles during viral infections. Like other TRIM proteins, TRIM7 can regulate the IFN pathway via the regulation of the cytosolic receptors RIG-I or MDA-5, which promote the production of type I interferons (IFN-I) and antiviral immune responses. Alternatively, under certain infectious conditions, TRIM7 can negatively regulate IFN-I signaling, resulting in increased virus replication. A growing body of evidence has also shown that TRIM7 can, in some cases, ubiquitinate viral proteins to promote viral replication and pathogenesis, while in other cases it can promote degradation of viral proteins through the proteasome, reducing virus infection. TRIM7 can also regulate the host inflammatory response and modulate the production of inflammatory cytokines, which can lead to detrimental inflammation. TRIM7 can also protect the host during infection by reducing cellular apoptosis. Here, we discuss the multiple functions of TRIM7 during viral infections and its potential as a therapeutic target.


Asunto(s)
Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas , Virosis , Replicación Viral , Humanos , Virosis/inmunología , Virosis/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas de Motivos Tripartitos/metabolismo , Proteínas de Motivos Tripartitos/genética , Animales , Ubiquitinación , Transducción de Señal , Interferón Tipo I/metabolismo , Interferón Tipo I/inmunología , Inmunidad Innata , Proteínas Virales/metabolismo , Proteínas Virales/genética , Proteínas Virales/inmunología , Interacciones Huésped-Patógeno/inmunología
5.
bioRxiv ; 2024 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-38948778

RESUMEN

SARS-CoV-2 is a highly transmissible virus that causes COVID-19 disease. Mechanisms of viral pathogenesis include excessive inflammation and viral-induced cell death, resulting in tissue damage. We identified the host E3-ubiquitin ligase TRIM7 as an inhibitor of apoptosis and SARS-CoV-2 replication via ubiquitination of the viral membrane (M) protein. Trim7 -/- mice exhibited increased pathology and virus titers associated with epithelial apoptosis and dysregulated immune responses. Mechanistically, TRIM7 ubiquitinates M on K14, which protects cells from cell death. Longitudinal SARS-CoV-2 sequence analysis from infected patients revealed that mutations on M-K14 appeared in circulating variants during the pandemic. The relevance of these mutations was tested in a mouse model. A recombinant M-K14/K15R virus showed reduced viral replication, consistent with the role of K15 in virus assembly, and increased levels of apoptosis associated with the loss of ubiquitination on K14. TRIM7 antiviral activity requires caspase-6 inhibition, linking apoptosis with viral replication and pathology.

6.
Annu Rev Pathol ; 18: 181-203, 2023 01 24.
Artículo en Inglés | MEDLINE | ID: mdl-36151059

RESUMEN

Zika virus (ZIKV) is an emerging virus from the Flaviviridae family that is transmitted to humans by mosquito vectors and represents an important health problem. Infections in pregnant women are of major concern because of potential devastating consequences during pregnancy and have been associated with microcephaly in newborns. ZIKV has a unique ability to use the host machinery to promote viral replication in a tissue-specific manner, resulting in characteristic pathological disorders. Recent studies have proposed that the host ubiquitin system acts as a major determinant of ZIKV tropism by providing the virus with an enhanced ability to enter new cells. In addition, ZIKV has developed mechanisms to evade the host immune response, thereby allowing the establishment of viral persistence and enhancing viral pathogenesis. We discuss recent reports on the mechanisms used by ZIKV to replicate efficiently, and we highlight potential new areas of research for the development of therapeutic approaches.


Asunto(s)
Microcefalia , Infección por el Virus Zika , Virus Zika , Recién Nacido , Animales , Femenino , Humanos , Embarazo , Infección por el Virus Zika/complicaciones , Infección por el Virus Zika/tratamiento farmacológico , Replicación Viral
7.
bioRxiv ; 2023 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-37503276

RESUMEN

Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the co-factor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity that correlated with reduced replication of infectious EBOV. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity.

8.
Sci Transl Med ; 15(699): eabq6517, 2023 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-37285402

RESUMEN

Zika virus (ZIKV) infection during pregnancy causes severe developmental defects in newborns, termed congenital Zika syndrome (CZS). Factors contributing to a surge in ZIKV-associated CZS are poorly understood. One possibility is that ZIKV may exploit the antibody-dependent enhancement of infection mechanism, mediated by cross-reactive antibodies from prior dengue virus (DENV) infection, which may exacerbate ZIKV infection during pregnancy. In this study, we investigated the impact of prior DENV infection or no DENV infection on ZIKV pathogenesis during pregnancy in a total of four female common marmosets with five or six fetuses per group. The results showed that negative-sense viral RNA copies increased in the placental and fetal tissues of DENV-immune dams but not in DENV-naïve dams. In addition, viral proteins were prevalent in endothelial cells, macrophages, and neonatal Fc receptor-expressing cells in the placental trabeculae and in neuronal cells in the brains of fetuses from DENV-immune dams. DENV-immune marmosets maintained high titers of cross-reactive ZIKV-binding antibodies that were poorly neutralizing, raising the possibility that these antibodies might be involved in the exacerbation of ZIKV infection. These findings need to be verified in a larger study, and the mechanism involved in the exacerbation of ZIKV infection in DENV-immune marmosets needs further investigation. However, the results suggest a potential negative impact of preexisting DENV immunity on subsequent ZIKV infection during pregnancy in vivo.


Asunto(s)
Virus del Dengue , Dengue , Infección por el Virus Zika , Virus Zika , Animales , Femenino , Embarazo , Callithrix , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Células Endoteliales , Placenta , Reacciones Cruzadas
9.
Cell Rep ; 38(10): 110434, 2022 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-35263596

RESUMEN

Type I interferons (IFN-I) are essential to establish antiviral innate immunity. Unanchored (or free) polyubiquitin (poly-Ub) has been shown to regulate IFN-I responses. However, few unanchored poly-Ub interactors are known. To identify factors regulated by unanchored poly-Ub in a physiological setting, we developed an approach to isolate unanchored poly-Ub from lung tissue. We identified the RNA helicase DHX16 as a potential pattern recognition receptor (PRR). Silencing of DHX16 in cells and in vivo diminished IFN-I responses against influenza virus. These effects extended to members of other virus families, including Zika and SARS-CoV-2. DHX16-dependent IFN-I production requires RIG-I and unanchored K48-poly-Ub synthesized by the E3-Ub ligase TRIM6. DHX16 recognizes a signal in influenza RNA segments that undergo splicing and requires its RNA helicase motif for direct, high-affinity interactions with specific viral RNAs. Our study establishes DHX16 as a PRR that partners with RIG-I for optimal activation of antiviral immunity requiring unanchored poly-Ub.


Asunto(s)
Proteína 58 DEAD Box , Interferón Tipo I , ARN Helicasas , ARN Viral , Receptores Inmunológicos , Infección por el Virus Zika , Virus Zika , COVID-19 , Proteína 58 DEAD Box/inmunología , Humanos , Inmunidad Innata , Interferón Tipo I/inmunología , ARN Helicasas/inmunología , Receptores Inmunológicos/inmunología , SARS-CoV-2 , Proteínas de Motivos Tripartitos , Virus Zika/genética , Infección por el Virus Zika/inmunología
10.
Viruses ; 13(3)2021 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-33652634

RESUMEN

Ubiquitination of proteins is a post-translational modification process with many different cellular functions, including protein stability, immune signaling, antiviral functions and virus replication. While ubiquitination of viral proteins can be used by the host as a defense mechanism by destroying the incoming pathogen, viruses have adapted to take advantage of this cellular process. The ubiquitin system can be hijacked by viruses to enhance various steps of the replication cycle and increase pathogenesis. Emerging viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), flaviviruses like Zika and dengue, as well as highly pathogenic viruses like Ebola and Nipah, have the ability to directly use the ubiquitination process to enhance their viral-replication cycle, and evade immune responses. Some of these mechanisms are conserved among different virus families, especially early during virus entry, providing an opportunity to develop broad-spectrum antivirals. Here, we discuss the mechanisms used by emergent viruses to exploit the host ubiquitin system, with the main focus on the role of ubiquitin in enhancing virus replication.


Asunto(s)
Ubiquitina/metabolismo , Virosis/metabolismo , Replicación Viral , Virus/metabolismo , Evasión Inmune , Ubiquitinación , Proteínas Virales/metabolismo , Ensamble de Virus , Virosis/inmunología , Virosis/virología , Internalización del Virus , Liberación del Virus , Virus/clasificación , Virus/inmunología , Virus/patogenicidad
11.
Curr Clin Microbiol Rep ; 7(4): 101-114, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32837832

RESUMEN

PURPOSE OF REVIEW: Tripartite motif (TRIM) proteins are a large group of E3 ubiquitin ligases involved in different cellular functions. Of special interest are their roles in innate immunity, inflammation, and virus replication. We discuss novel roles of TRIM proteins during virus infections that lead to increased pathogenicity. RECENT FINDINGS: TRIM proteins regulate different antiviral and inflammatory signaling pathways, mostly by promoting ubiquitination of important factors including pattern recognition receptors, adaptor proteins, kinases, and transcription factors that are involved in type I interferon and NF-κB pathways. Therefore, viruses have developed mechanisms to target TRIMs for immune evasion. New evidence is emerging indicating that viruses have the ability to directly use TRIMs and the ubiquitination process to enhance the viral replication cycle and cause increased pathogenesis. A new report on TRIM7 also highlights the potential pro-viral role of TRIMs via ubiquitination of viral proteins and suggests a novel mechanism by which ubiquitination of virus envelope protein may provide determinants of tissue and species tropism. SUMMARY: TRIM proteins have important functions in promoting host defense against virus infection; however, viruses have adapted to evade TRIM-mediated immune responses and can hijack TRIMs to ultimately increase virus pathogenesis. Only by understanding specific TRIM-virus interactions and by using more in vivo approaches can we learn how to harness TRIM function to develop therapeutic approaches to reduce virus pathogenesis.

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