SARS-CoV-2 NSP12 Protein Is Not an Interferon-ß Antagonist.

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is bringing an unprecedented health crisis to the world. To date, our understanding of the interaction between SARS-CoV-2 and host innate immunity is still limited. Previous studies reported that SARS-CoV-2 nonstructural protein 12 (NSP12) was able to suppress interferon-ß (IFN-ß) activation in IFN-ß promoter luciferase reporter assays, which provided insights into the pathogenesis of COVID-19. In this study, we demonstrated that IFN-ß promoter-mediated luciferase activity was reduced during coexpression of NSP12. However, we could show NSP12 did not affect IRF3 or NF-κB activation. Moreover, IFN-ß production induced by Sendai virus (SeV) infection or other stimulus was not affected by NSP12 at mRNA or protein level. Additionally, the type I IFN signaling pathway was not affected by NSP12, as demonstrated by the expression of interferon-stimulated genes (ISGs). Further experiments revealed that different experiment systems, including protein tags and plasmid backbones, could affect the readouts of IFN-ß promoter luciferase assays. In conclusion, unlike as previously reported, our study showed SARS-CoV-2 NSP12 protein is not an IFN-ß antagonist. It also rings the alarm on the general usage of luciferase reporter assays in studying SARS-CoV-2. IMPORTANCE Previous studies investigated the interaction between SARS-CoV-2 viral proteins and interferon signaling and proposed that several SARS-CoV-2 viral proteins, including NSP12, could suppress IFN-ß activation. However, most of these results were generated from IFN-ß promoter luciferase reporter assay and have not been validated functionally. In our study, we found that, although NSP12 could suppress IFN-ß promoter luciferase activity, it showed no inhibitory effect on IFN-ß production or its downstream signaling. Further study revealed that contradictory results could be generated from different experiment systems. On one hand, we demonstrated that SARS-CoV-2 NSP12 could not suppress IFN-ß signaling. On the other hand, our study suggests that caution needs to be taken with the interpretation of SARS-CoV-2-related luciferase assays.

Matrix metalloproteinase 9 facilitates Zika virus invasion of the testis by modulating the integrity of the blood-testis barrier.

Zika virus (ZIKV) is a unique flavivirus with high tropism to the testes. ZIKV can persist in human semen for months and can cause testicular damage in male mice. However, the mechanisms through which ZIKV enters the testes remain unclear. In this study, we revealed that matrix metalloproteinase 9 (MMP9) was upregulated by ZIKV infection in cell culture and in A129 mice. Furthermore, using an in vitro Sertoli cell barrier model and MMP9-/- mice, we found that ZIKV infection directly affected the permeability of the blood-testis barrier (BTB), and knockout or inhibition of MMP9 reduced the effects of ZIKV on the Sertoli cell BTB, highlighting its role in ZIKV-induced disruption of the BTB. Interestingly, the protein levels of MMP9 were elevated by ZIKV nonstructural protein 1 (NS1) in primary mouse Sertoli cells (mSCs) and other cell lines. Moreover, the interaction between NS1 and MMP9 induced the K63-linked polyubiquitination of MMP9, which enhanced the stability of MMP9. The upregulated MMP9 level led to the degradation of essential proteins involved in the maintenance of the BTB, such as tight junction proteins (TJPs) and type Ⅳ collagens. Collectively, we concluded that ZIKV infection promoted the expression of MMP9 which was further stabilized by NS1 induced K63-linked polyubiquitination to affect the TJPs/ type Ⅳ collagen network, thereby disrupting the BTB and facilitating ZIKV entry into the testes.

ZIKV viral proteins and their roles in virus-host interactions.

The re-emergence of Zika virus (ZIKV) and its associated neonatal microcephaly and Guillain-Barré syndrome have led the World Health Organization to declare a global health emergency. Until today, many related studies have successively reported the role of various viral proteins of ZIKV in the process of ZIKV infection and pathogenicity. These studies have provided significant insights for the treatment and prevention of ZIKV infection. Here we review the current research advances in the functional characterization of the interactions between each ZIKV viral protein and its host factors.

Erratum to: Tick virome diversity in Hubei Province, China, and the influence of host ecology.

[This corrects the article DOI: 10.1093/ve/veab089.].

Tick virome diversity in Hubei Province, China, and the influence of host ecology.

Ticks are important vector hosts of pathogens which cause human and animal diseases worldwide. Diverse viruses have been discovered in ticks; however, little is known about the ecological factors that affect the tick virome composition and evolution. Herein, we employed RNA sequencing to study the virome diversity of the Haemaphysalis longicornis and Rhipicephalus microplus ticks sampled in Hubei Province in China. Twelve RNA viruses with complete genomes were identified, which belonged to six viral families: Flaviviridae, Matonaviridae, Peribunyaviridae, Nairoviridae, Phenuiviridae, and Rhabdoviridae. These viruses showed great diversity in their genome organization and evolution, four of which were proposed to be novel species. The virome diversity and abundance of R. microplus ticks fed on cattle were evidently high. Further ecological analyses suggested that host species and feeding status may be key factors affecting the tick virome structure. This study described a number of novel viral species and variants from ticks and, more importantly, provided insights into the ecological factors shaping the virome structures of ticks, although it clearly warrants further investigation.

Rearrangement of Actin Cytoskeleton by Zika Virus Infection Facilitates Blood-Testis Barrier Hyperpermeability.

In recent years, various serious diseases caused by Zika virus (ZIKV) have made it impossible to be ignored. Confirmed existence of ZIKV in semen and sexually transmission of ZIKV suggested that it can break the blood-testis barrier (BTB), or Sertoli cell barrier (SCB). However, little is known about the underlying mechanism. In this study, interaction between actin, an important component of the SCB, and ZIKV envelope (E) protein domain III (EDIII) was inferred from co-immunoprecipitation (Co-IP) liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Confocal microscopy confirmed the role of actin filaments (F-actin) in ZIKV infection, during which part of the stress fibers, the bundles that constituted by paralleled actin filaments, were disrupted and presented in the cell periphery. Colocalization of E and reorganized actin filaments in the cell periphery of transfected Sertoli cells suggests a participation of ZIKV E protein in ZIKV-induced F-actin rearrangement. Perturbation of F-actin by cytochalasin D (CytoD) or Jasplakinolide (Jas) enhanced the infection of ZIKV. More importantly, the transepithelial electrical resistance (TEER) of an in vitro mouse SCB (mSCB) model declined with the progression of ZIKV infection or overexpression of E protein. Co-IP and confocal microscopy analyses revealed that the interaction between F-actin and tight junction protein ZO-1 was reduced after ZIKV infection or E protein overexpression, highlighting the role of E protein in ZIKV-induced disruption of the BTB. We conclude that the interaction between ZIKV E and F-actin leads to the reorganization of F-actin network, thereby compromising BTB integrity.

NS5 Conservative Site Is Required for Zika Virus to Restrict the RIG-I Signaling.

During host-virus co-evolution, cells develop innate immune systems to inhibit virus invasion, while viruses employ strategies to suppress immune responses and maintain infection. Here, we reveal that Zika virus (ZIKV), a re-emerging arbovirus causing public concerns and devastating complications, restricts host immune responses through a distinct mechanism. ZIKV nonstructural protein 5 (NS5) interacts with the host retinoic acid-inducible gene I (RIG-I), an essential signaling molecule for defending pathogen infections. NS5 subsequently represses K63-linked polyubiquitination of RIG-I, attenuates the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3), and inhibits the expression and production of interferon-ß (IFN-ß), thereby restricting the RIG-I signaling pathway. Interestingly, we demonstrate that the methyltransferase (MTase) domain of NS5 is required for the repression of RIG-I ubiquitination, IRF3 activation, and IFN-ß production. Detailed studies further reveal that the conservative active site D146 of NS5 is critical for the suppression of the RIG-I signaling. Therefore, we uncover an essential role of NS5 conservative site D146 in ZIKV-mediated repression of innate immune system, illustrate a distinct mechanism by which ZIKV evades host immune responses, and discover a potential target for anti-viral infection.