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1.
J Virol ; 98(6): e0170523, 2024 Jun 13.
Artículo en Inglés | MEDLINE | ID: mdl-38742902

RESUMEN

Long non-coding RNAs (lncRNAs) represent a new group of host factors involved in viral infection. Current study identified an intergenic lncRNA, LINC08148, as a proviral factor of Zika virus (ZIKV) and Dengue virus 2 (DENV2). Knockout (KO) or silencing of LINC08148 decreases the replication of ZIKV and DENV2. LINC08148 mainly acts at the endocytosis step of ZIKV but at a later stage of DENV2. RNA-seq analysis reveals that LINC08148 knockout downregulates the transcription levels of five endocytosis-related genes including AP2B1, CHMP4C, DNM1, FCHO1, and Src. Among them, loss of Src significantly decreases the uptake of ZIKV. Trans-complementation of Src in the LINC08148KO cells largely restores the caveola-mediated endocytosis of ZIKV, indicating that the proviral effect of LINC08148 is exerted through Src. Finally, LINC08148 upregulates the Src transcription through associating with its transcription factor SP1. This work establishes an essential role of LINC08148 in the ZIKV entry, underscoring a significance of lncRNAs in the viral infection. IMPORTANCE: Long non-coding RNAs (lncRNAs), like proteins, participate in viral infection. However, functions of most lncRNAs remain unknown. In this study, we performed a functional screen based on microarray data and identified a new proviral lncRNA, LINC08148. Then, we uncovered that LINC08148 is involved in the caveola-mediated endocytosis of ZIKV, rather than the classical clathrin-mediated endocytosis. Mechanistically, LINC08148 upregulates the transcription of Src, an initiator of caveola-mediated endocytosis, through binding to its transcription factor SP1. This study identifies a new lncRNA involved in the ZIKV infection, suggesting lncRNAs and cellular proteins are closely linked and cooperate to regulate viral infection.


Asunto(s)
Endocitosis , ARN Largo no Codificante , Internalización del Virus , Infección por el Virus Zika , Virus Zika , ARN Largo no Codificante/metabolismo , ARN Largo no Codificante/genética , Virus Zika/genética , Virus Zika/fisiología , Humanos , Infección por el Virus Zika/virología , Infección por el Virus Zika/metabolismo , Infección por el Virus Zika/genética , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp1/genética , Caveolas/metabolismo , Animales , Replicación Viral , Regulación hacia Arriba , Virus del Dengue/fisiología , Virus del Dengue/genética , Chlorocebus aethiops , Células HEK293 , Células Vero , Familia-src Quinasas/metabolismo , Familia-src Quinasas/genética
2.
Nat Commun ; 15(1): 296, 2024 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-38177122

RESUMEN

Cytoskeleton is extensively recruited by flaviviruses for their infection. In this study, we uncovered an essential role of a nuclear membrane protein, SAD1/UNC84 domain protein 2 (SUN2) linking cytoskeleton and nucleoskeleton in the flavivirus replication. CRISPR/Cas9-mediated knockout of SUN2, but not SUN1, significantly reduces the replication of Zika virus (ZIKV), dengue virus (DENV), and Japanese encephalitis virus (JEV). In contrast, SUN2 does not affect the infection of non-flaviviridae RNA viruses. All three regions of SUN2 are required for its proviral effect. Mechanistically, SUN2 facilitates rearrangement of cytoskeleton and formation of replication organelles induced by viral infection, and hence promotes viral RNA synthesis. SUN2 is required for the interaction between cytoskeleton actin and ZIKV nonstructural protein 1 (NS1). Expression of dominant negative Nesprin-1 and Nesprin-2, which connect SUN2 to cytoskeleton proteins, alleviates the interaction between actin and NS1 and reduces viral replication levels. In a neonatal mouse infection model, SUN2 knockout dramatically alleviates the in vivo ZIKV replication and development of neuropathology. This work elucidates that recruitment of cytoskeleton proteins by flavivirus is coordinated by nuclear membrane proteins SUN2 and Nesprins, providing evidence for a link between nuclear membrane proteins and flavivirus infection.


Asunto(s)
Proteínas de la Membrana , Infección por el Virus Zika , Virus Zika , Animales , Ratones , Actinas/metabolismo , Citoesqueleto/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas no Estructurales Virales/química , Replicación Viral , Virus Zika/genética , Virus Zika/fisiología
3.
Microbiol Spectr ; 10(5): e0105622, 2022 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-36000889

RESUMEN

Infection by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has posed a severe threat to global public health. The current study revealed that several inhibitors of protein kinases C (PKCs) possess protective activity against SARS-CoV-2 infection. Four pan-PKC inhibitors, Go 6983, bisindolylmaleimide I, enzastaurin, and sotrastaurin, reduced the replication of a SARS-CoV-2 replicon in both BHK-21 and Huh7 cells. A PKCδ-specific inhibitor, rottlerin, was also effective in reducing viral infection. The PKC inhibitors acted at an early step of SARS-CoV-2 infection. Finally, PKC inhibitors blocked the replication of wild-type SARS-CoV-2 in ACE2-expressing A549 cells. Our work highlights the importance of the PKC signaling pathway in infection by SARS-CoV-2 and provides evidence that PKC-specific inhibitors are potential therapeutic agents against SARS-CoV-2. IMPORTANCE There is an urgent need for effective therapeutic drugs to control the pandemic caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). We found that several inhibitors of protein kinases C (PKCs) dramatically decrease the replication of SARS-CoV-2 in cultured cells. These PKC inhibitors interfere with an early step of viral infection. Therefore, the rapid and prominent antiviral effect of PKC inhibitors underscores that they are promising antiviral agents and suggests that PKCs are important host factors involved in infection by SARS-CoV-2.


Asunto(s)
Antivirales , Proteína Quinasa C , SARS-CoV-2 , Humanos , Enzima Convertidora de Angiotensina 2 , Antivirales/farmacología , Células Cultivadas , Proteína Quinasa C/farmacología , SARS-CoV-2/efectos de los fármacos , Tratamiento Farmacológico de COVID-19
4.
Virol Sin ; 37(5): 685-694, 2022 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-35934227

RESUMEN

Infection of Zika virus (ZIKV) may cause microcephaly and other neurological disorders, while no vaccines and drugs are available. Our study revealed that rottlerin confers a broad antiviral activity against several enveloped viruses, including ZIKV, vesicular stomatitis virus, and herpes simplex virus, but not against two naked viruses (enterovirus 71 and encephalomyocarditis virus). Rottlerin does not have a direct virucidal effect on the virions, and its antiviral effect is independent of its regulation on PKCδ or ATP. Both pretreatment and post-treatment of rottlerin effectively reduce the viral replication of ZIKV. The pretreatment of rottlerin disturbs the endocytosis of enveloped viruses, while the post-treatment of rottlerin acts at a late stage through disturbing the maturation of ZIKV. Importantly, administration of rottlerin in neonatal mice significantly decreased the ZIKV replication in vivo, and alleviated the neurological symptoms caused by ZIKV. Our work suggests that rottlerin exerts an antiviral activity at two distinct steps of viral infection, and can be potentially developed as a prophylactic and therapeutic agent.


Asunto(s)
Infección por el Virus Zika , Virus Zika , Acetofenonas , Adenosina Trifosfato/farmacología , Adenosina Trifosfato/uso terapéutico , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Benzopiranos , Ratones , Replicación Viral
5.
J Cell Sci ; 135(6)2022 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-35112703

RESUMEN

We performed an unbiased whole-genome CRISPR/Cas9 screen in A549 cells to identify potential regulators involved in cell death triggered by double-stranded RNA (dsRNA). Of several top candidate genes, we identified the RNA-binding gene ELAV like protein 1 (256529), which encodes the protein Hu antigen R (HuR). Depletion of HuR led to less cell death induced by dsRNA. HuR is mainly involved in apoptosis, and all of its RNA recognition motifs are essential for its pro-apoptotic function. We further showed that the HuR depletion had no influence on the mRNA level of the anti-apoptotic gene BCL2, but instead that HuR downregulates BCL2 translation in a cap-independent way. Polysome fractionation studies showed that HuR retarded the BCL2 mRNA in the non-translating pool of polysomes. Moreover, protection from dsRNA-induced apoptosis by HuR depletion required the presence of BCL2, indicating that the pro-apoptotic function of HuR is executed by suppressing BCL2. Consistent with this, HuR regulated apoptosis induced by infection of encephalomyocarditis or Semliki Forest virus. Collectively, our work identified a suite of proteins that regulate dsRNA-induced cell death, and elucidated the mechanism by which HuR acts as a pro-apoptotic factor.


Asunto(s)
Proteína 1 Similar a ELAV , ARN Bicatenario , Apoptosis/genética , Proteínas ELAV/genética , Proteínas ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Proteína 1 Similar a ELAV/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Bicatenario/genética , ARN Mensajero/genética
6.
J Cell Sci ; 135(1)2022 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-34859815

RESUMEN

Apoptosis is an important cellular response to viral infection. In this study, we identified activating molecule in Beclin1-regulated autophagy protein 1 (AMBRA1) as a positive regulator of apoptosis triggered by double-stranded (ds)RNA. Depletion of AMBRA1 by gene editing significantly reduced dsRNA-induced apoptosis, which was largely restored by trans-complementation of AMBRA1. Mechanistically, AMBRA1 interacts with mitochondrial antiviral-signaling protein (MAVS), a key mitochondrial adaptor in the apoptosis pathway induced by dsRNA and viral infection. Further co-immunoprecipitation analysis demonstrated that the mitochondrial localization of MAVS was essential for their interaction. The impact of AMBRA1 on dsRNA-induced apoptosis relied on the presence of MAVS and caspase-8. AMBRA1 was involved in the stabilization of MAVS through preventing its dsRNA-induced proteasomal degradation. Consistently, AMBRA1 upregulated the apoptosis induced by Semliki Forest virus infection. Taken together, our work illustrated a role for AMBRA1 in virus-induced apoptosis through interacting with and stabilizing MAVS.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Apoptosis , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/genética , Autofagia , Beclina-1 , ARN Bicatenario/genética
7.
J Innate Immun ; 13(3): 179-193, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33626545

RESUMEN

Expression of host noncoding RNAs and coding mRNAs is significantly altered by viral infection. In the current study, we screened the transcriptional profile of human lung epithelial A549 cells infected with Zika virus (ZIKV) by microarray assay. Seventy-nine long noncoding RNAs (lncRNAs) and 140 mRNAs were differentially expressed (DE). The bioinformatics analysis revealed that the mRNAs adjacent to the DE lncRNAs were closely related to the host responses to viral infection. We selected 7 lncRNAs from the top 50 hits for validation. The quantitative real-time PCR data confirmed that expression of selected lncRNAs was induced by ZIKV infection. Moreover, the expression of 7 lncRNAs was induced by infection of dengue virus, Japanese encephalitis virus, or vesicular stomatitis virus, or by treatment of poly(I:C) and IFN-ß. Furthermore, loss of innate immune adaptor IPS-1 or receptor IFNAR1 resulted in lower induction levels of several lncRNAs by ZIKV. Overexpression of 3 lncRNAs (RPL27-OT1, OASL-IT1, and REC8-OT3) reduced the virus yields of ZIKV. Knockout of OASL-IT1 significantly enhanced ZIKV replication. In OASL-IT1 knockout cells, the levels of interferons (IFNs) and the activation of 3 innate immune signaling pathways triggered by ZIKV were dramatically reduced. Collectively, our work found a positive feedback loop in the IFN system, in which IFNs and OASL-IT1 regulate each other, thereby promoting establishment of antiviral defense.


Asunto(s)
ARN Largo no Codificante/genética , Mucosa Respiratoria/inmunología , Virosis/inmunología , Infección por el Virus Zika/inmunología , Virus Zika/fisiología , Células A549 , Biología Computacional , Retroalimentación Fisiológica , Técnicas de Silenciamiento del Gen , Humanos , Inmunidad Innata , Interferón beta/metabolismo , Poli I-C/inmunología , Transducción de Señal/inmunología , Replicación Viral
8.
J Biol Chem ; 294(48): 18168-18180, 2019 11 29.
Artículo en Inglés | MEDLINE | ID: mdl-31636123

RESUMEN

Zika virus (ZIKV) is a mosquito-borne flavivirus that has emerged as a threat to global health. The family of adenosine deaminases acting on dsRNA (ADARs) are human host factors important for the genetic diversity and evolution of ZIKV. Here, we further investigated the role of ADAR1 in ZIKV replication by utilizing CRISPR/Cas9-based gene editing and RNAi-based gene knockdown techniques. Both ADAR1 knockout and knockdown significantly reduced ZIKV RNA synthesis, protein levels, and viral titers in several human cell lines. Trans-complementation with the full-length ADAR1 form p150 or the shorter form p110 lacking the Zα domain restored viral replication levels suppressed by the ADAR1 knockout. Moreover, we observed that the nuclear p110 form was redistributed to the cytoplasm in response to ZIKV infection. ADAR1 was not involved in viral entry but promoted viral protein translation by impairing ZIKV-induced activation of protein kinase regulated by dsRNA (PKR). Of note, the RNA-editing activity of ADAR1 was not required to promote ZIKV replication. We also found that the proviral role of ADAR1 was partially mediated through its ability to suppress IFN production and PKR activation. Our work identifies ADAR1 as a proviral factor involved in ZIKV replication, suggesting that ADAR1 could be a potential antiviral target.


Asunto(s)
Adenosina Desaminasa/metabolismo , Biosíntesis de Proteínas/fisiología , Proteínas de Unión al ARN/metabolismo , Proteínas Virales/biosíntesis , Replicación Viral/fisiología , Virus Zika/fisiología , eIF-2 Quinasa/metabolismo , Células A549 , Adenosina Desaminasa/genética , Animales , Chlorocebus aethiops , Activación Enzimática , Células HEK293 , Humanos , Proteínas de Unión al ARN/genética , Células Vero , Proteínas Virales/genética , eIF-2 Quinasa/genética
9.
Virology ; 529: 91-100, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30684694

RESUMEN

Zika virus (ZIKV) is an emerging arbovirus and its infection associates with neurologic diseases. Whether heparan sulfate (HS), an attachment factor for many viruses, plays a role in the ZIKV infection remains controversial. Our study generated several HS biosynthesis-deficient cell clones by disrupting SLC35B2, B3GAT3, or B4GALT7 gene using the CRISPR/Cas9 system. The HS deficiency did not affect the viral attachment and internalization of ZIKV, but reduced the attachment of Dengue virus (DENV) 2. The early RNA and protein levels of ZIKV and DENV2 were impaired in the HS deficient cells, while the viral yields were not accordingly reduced. Our data further showed that HS promoted the cell death induced by virus infection, and inhibition of cell death significantly increased the viral replication of ZIKV and DENV2. Collectively, our study described an unexpected role of HS in the viral attachment, replication and cell death induced by ZIKV.


Asunto(s)
Muerte Celular , Heparitina Sulfato/metabolismo , Internalización del Virus , Replicación Viral/fisiología , Virus Zika/fisiología , Animales , Línea Celular , Humanos , Interferón beta , Regulación hacia Arriba , Virus Zika/genética
10.
Artículo en Inglés | MEDLINE | ID: mdl-32039055

RESUMEN

Zika virus (ZIKV) is an emerging arthropod-borne virus and belongs to the Flaviviridae family. The infection of ZIKV has become the global health crisis because of its rapid spread and association with severe neurological disorders, including congenital microcephaly and Guillain-Barre Syndrome. To identify host factors contributing to ZIKV pathogenesis, transcriptomic landscape in ZIKV-infected cells was examined with mRNA microarray analysis and we observed that the expression of hydroxycarboxylic acid receptor 2 (HCAR2) could be significantly induced by ZIKV infection. By utilizing two IRE1 inhibitors and XBP1-specific shRNAs, we revealed that the up-regulation of HCAR2 expression induced by ZIKV was dependent on the IRE1-XBP1 pathway. Through the CRISPR/Cas9 system, we generated HCAR2-deficient cell clones in two cell types (human lung carcinoma epithelial A549 cell and human hepatoma Huh7.5 cell). We found that the depletion of HCAR2 significantly increased the replication level of ZIKV, including RNA levels, protein expression levels, and viral titers. In addition, our data demonstrated that the antiviral effect of HCAR2 was not involved in viral entry process and was not dependent on its antilipolytic effect on nicotinic acid/HCAR2-mediated signaling pathway. Taken together, our results indicated that HCAR2 could function as a restriction factor in control of ZIKV replication, potentially providing a novel molecular target for anti-ZIKV therapeutics.


Asunto(s)
Endorribonucleasas/metabolismo , Factores Inmunológicos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Proteína 1 de Unión a la X-Box/metabolismo , Infección por el Virus Zika/inmunología , Línea Celular , Edición Génica , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Análisis por Micromatrices , Mapas de Interacción de Proteínas , Replicación Viral , Virus Zika/crecimiento & desarrollo
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