RESUMO
African horse sickness virus (AHSV) non-structural protein NS4 is a nucleocytoplasmic protein that is expressed in the heart, lung, and spleen of infected horses, binds dsDNA, and colocalizes with promyelocytic leukemia nuclear bodies (PML-NBs). The aim of this study was to investigate the role of AHSV NS4 in viral replication, virulence and the host immune response. Using a reverse genetics-derived virulent strain of AHSV-5 and NS4 deletion mutants, we showed that knockdown of NS4 expression has no impact in cell culture, but results in virus attenuation in infected horses. RNA sequencing (RNA-seq) was used to investigate the transcriptional response in these horses, to see how the lack of NS4 mediates the transition of the virus from virulent to attenuated. The presence of NS4 was shown to result in a 24 hour (h) delay in the transcriptional activation of several immune system processes compared to when the protein was absent. Included in these processes were the RIG-I-like, Toll-like receptor, and JAK-STAT signaling pathways, which are key pathways involved in innate immunity and the antiviral response. Thus, it was shown that AHSV NS4 suppresses the host innate immune transcriptional response in the early stages of the infection cycle. We investigated whether AHSV NS4 affects the innate immune response by impacting the JAK-STAT signaling pathway specifically. Using confocal laser scanning microscopy (CLSM) we showed that AHSV NS4 disrupts JAK-STAT signaling by interfering with the phosphorylation and/or translocation of STAT1 and pSTAT1 into the nucleus. Overall, these results showed that AHSV NS4 is a key virulence factor in horses and allows AHSV to overcome host antiviral responses in order to promote viral replication and spread.
Assuntos
Vírus da Doença Equina Africana , Doença Equina Africana , Vírus da Doença Equina Africana/genética , Animais , Cavalos , Transdução de Sinais , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
We announce the complete consensus genome sequence of 27 African horse sickness viruses, representing all nine African horse sickness virus (AHSV) serotypes from historical and recent isolates collected over a 76-year period (1933 to 2009). The data set includes the sequence of the virulent Office International des Epizooties AHSV reference strains which are not adapted to cell culture.
RESUMO
African horsesickness (AHS) is a devastating disease of horses. The disease is caused by the double-stranded RNA-containing African horsesickness virus (AHSV). Using electron cryomicroscopy and three-dimensional image reconstruction, we determined the architecture of an AHSV serotype 4 (AHSV-4) reference strain. The structure revealed triple-layered AHS virions enclosing the segmented genome and transcriptase complex. The innermost protein layer contains 120 copies of VP3, with the viral polymerase, capping enzyme, and helicase attached to the inner surface of the VP3 layer on the 5-fold axis, surrounded by double-stranded RNA. VP7 trimers form a second, T=13 layer on top of VP3. Comparative analyses of the structures of bluetongue virus and AHSV-4 confirmed that VP5 trimers form globular domains and VP2 trimers form triskelions, on the virion surface. We also identified an AHSV-7 strain with a truncated VP2 protein (AHSV-7 tVP2) which outgrows AHSV-4 in culture. Comparison of AHSV-7 tVP2 to bluetongue virus and AHSV-4 allowed mapping of two domains in AHSV-4 VP2, and one in bluetongue virus VP2, that are important in infection. We also revealed a protein plugging the 5-fold vertices in AHSV-4. These results shed light on virus-host interactions in an economically important orbivirus to help the informed design of new vaccines.