LGP2 directly interacts with flavivirus NS5 RNA-dependent RNA polymerase and downregulates its pre-elongation activities.

LGP2 is a RIG-I-like receptor (RLR) known to bind and recognize the intermediate double-stranded RNA (dsRNA) during virus infection and to induce type-I interferon (IFN)-related antiviral innate immune responses. Here, we find that LGP2 inhibits Zika virus (ZIKV) and tick-borne encephalitis virus (TBEV) replication independent of IFN induction. Co-immunoprecipitation (Co-IP) and confocal immunofluorescence data suggest that LGP2 likely colocalizes with the replication complex (RC) of ZIKV by interacting with viral RNA-dependent RNA polymerase (RdRP) NS5. We further verify that the regulatory domain (RD) of LGP2 directly interacts with RdRP of NS5 by biolayer interferometry assay. Data from in vitro RdRP assays indicate that LGP2 may inhibit polymerase activities of NS5 at pre-elongation but not elongation stages, while an RNA-binding-defective LGP2 mutant can still inhibit RdRP activities and virus replication. Taken together, our work suggests that LGP2 can inhibit flavivirus replication through direct interaction with NS5 protein and downregulates its polymerase pre-elongation activities, demonstrating a distinct role of LGP2 beyond its function in innate immune responses.

Identification of novel anti-ZIKV drugs from viral-infection temporal gene expression profiles.

Zika virus (ZIKV) infections are typically asymptomatic but cause severe neurological complications (e.g. Guillain-Barré syndrome in adults, and microcephaly in newborns). There are currently no specific therapy or vaccine options available to prevent ZIKV infections. Temporal gene expression profiles of ZIKV-infected human brain microvascular endothelial cells (HBMECs) were used in this study to identify genes essential for viral replication. These genes were then used to identify novel anti-ZIKV agents and validated in publicly available data and functional wet-lab experiments. Here, we found that ZIKV effectively evaded activation of immune response-related genes and completely reprogrammed cellular transcriptional architectures. Knockdown of genes, which gradually upregulated during viral infection but showed distinct expression patterns between ZIKV- and mock infection, discovered novel proviral and antiviral factors. One-third of the 74 drugs found through signature-based drug repositioning and cross-reference with the Drug Gene Interaction Database (DGIdb) were known anti-ZIKV agents. In cellular assays, two promising antiviral candidates (Luminespib/NVP-AUY922, L-161982) were found to reduce viral replication without causing cell toxicity. Overall, our time-series transcriptome-based methods offer a novel and feasible strategy for antiviral drug discovery. Our strategies, which combine conventional and data-driven analysis, can be extended for other pathogens causing pandemics in the future.

Metabolic labeling of enterovirus 71 with quantum dots for the study of virus receptor usage.

Fluorescent labeling and dynamic tracking is a powerful tool for exploring virus infection mechanisms. However, for small-sized viruses, virus tracking studies are usually hindered by a lack of appropriate labeling methods that do not dampen virus yield or infectivity. Here, we report a universal strategy for labeling viruses with chemical dyes and Quantum dots (QDs). Enterovirus 71 (EV71) was produced in a cell line that stably expresses a mutant methionyl-tRNA synthetase (MetRS), which can charge azidonorleucine (ANL) to the methionine sites of viral proteins during translation. Then, the ANL-containing virus was easily labeled with DBCO-AF647 and DBCO-QDs. The labeled virus shows sufficient yield and no obvious decrease in infectivity and can be used for imaging the virus entry process. Using the labeled EV71, different functions of scavenger receptor class B, member 2 (SCARB2), and heparan sulfate (HS) in EV71 infection were comparatively studied. The cell entry process of a strong HS-binding EV71 strain was investigated by real-time dynamic visualization of EV71-QDs in living cells. Taken together, our study described a universal biocompatible virus labeling method, visualized the dynamic viral entry process, and reported details of the receptor usage of EV71.

Recovery of a Far-Eastern Strain of Tick-Borne Encephalitis Virus with a Full-Length Infectious cDNA Clone.

Tick-borne encephalitis virus (TBEV) is a pathogenic virus known to cause central nervous system (CNS) diseases in humans, and has become an increasing public health threat nowadays. The rates of TBEV infection in the endemic countries are increasing. However, there is no effective antiviral against the disease. This underscores the urgent need for tools to study the emergence and pathogenesis of TBEV and to accelerate the development of vaccines and antivirals. In this study, we reported an infectious cDNA clone of TBEV that was isolated in China (the WH2012 strain). A beta-globin intron was inserted in the coding region of nonstructural protein 1 (NS1) gene to improve the stability of viral genome in bacteria. In mammalian cells, the inserted intron was excised and spliced precisely, which did not lead to the generation of inserted mutants. High titers of infectious progeny viruses were generated after the transfection of the infectious clone. The cDNA-derived TBEV replicated efficiently, and caused typical cytopathic effect (CPE) and plaques in BHK-21 cells. In addition, the CPE and growth curve of cDNA-derived virus were similar to that of its parental isolate in cells. Together, we have constructed the first infectious TBEV cDNA clone in China, and the clone can be used to investigate the genetic determinants of TBEV virulence and disease pathogenesis, and to develop countermeasures against the virus.

In vivo imaging of Zika virus reveals dynamics of viral invasion in immune-sheltered tissues and vertical propagation during pregnancy.

Rationale: Zika virus (ZIKV) is a pathogenic virus known to cause a wide range of congenital abnormalities, including microcephaly, Guillain-Barre syndrome, meningoencephalitis, and other neurological complications, in humans. This study investigated the noninvasive detection of ZIKV infection in vivo, which is necessary for elucidating the virus's mechanisms of viral replication and pathogenesis, as well as to accelerate the development of anti-ZIKV therapeutic strategies. Methods: In this study, a recombinant ZIKV harbouring Nluc gene (ZIKV-Nluc) was designed, recovered, and purified. The levels of bioluminescence were directly correlated with viral loads in vitro and in vivo. The dynamics of ZIKV infection in A129 (interferon (IFN)-α/ß receptor deficient), AG6 (IFN-α/ß and IFN-γ receptor deficient), and C57BL/6 mice were characterized. Pregnant dams were infected with ZIKV-Nluc at E10 via intra footpad injection. Then, the pooled immune sera (anti-ZIKV neutralizing antibodies) #22-1 in ZIKV-Nluc virus-infected mice were visualized. Results: ZIKV-Nluc showed a high genetic stability and replicated well in cells with similar properties to the wild-type ZIKV (ZIKVwt). Striking bioluminescence signals were consistently observed in animal organs, including spleen, intestine, testis, uterus/ovary, and kidney. The ileocecal junction was found to be the crucial visceral target. Infection of pregnant dams with ZIKV-Nluc showed that ZIKV was capable of crossing the maternal-fetal barrier to infect the fetuses via vertical transmission. Furthermore, it was visualized that treatment with the pooled immune sera was found to greatly restrict the spread of the ZIKV-Nluc virus in mice. Conclusions: This study is the first to report the real-time noninvasive tracking of the progression of ZIKV invading immune-sheltered tissues and propagating vertically during pregnancy. The results demonstrate that ZIKV-Nluc represents a powerful tool for the study of the replication, dissemination, pathogenesis, and treatment of ZIKV in vitro and in vivo.

Baculovirus Surface Display of Zika Virus Envelope Protein Protects against Virus Challenge in Mouse Model.

Zika virus (ZIKV) is emerging as a significant pathogen worldwide and may cause severe neurological disorders such as fetal microcephaly and Guillain-Barre syndrome. No drug or listed vaccines are currently available for preventing ZIKV infection. As a major target of neutralizing, ZIKV envelop (E) protein usually used for vaccine development. Nevertheless, the immunogenicity of ZIKV envelop (E) protein expressed by baculovirus display system has never been assessed. In this study, we reported a new strategy for surface display of ZIKV E protein by a recombinant baculovirus vector derived from Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and assessed its immunogenicity in mice. We produced recombinant fusion ZIKV E protein linked with signal peptide (SP) and transmembrane domain (TM) of AcMNPV GP64. The results showed that the recombinant protein was easy to produce by baculovirus display system. BALB/c mice immunized with this recombinant E protein developed ZIKV specific serum antibodies. The anti-E protein sera from the mice were able to effectively neutralize ZIKV in vitro. More importantly, AG6 (IFN-α/ß and IFN-γ receptor deficient) mice immunized with recombinant E protein were protected against lethal ZIKV challenge. Together, these findings demonstrated that the recombinant E protein displayed by baculovirus can be conveniently prepared and displayed good immunogenicity in immunized mice. It is a promising practical approach for prompting the development of vaccine and related immunology research.

Zika virus promotes CCN1 expression via the CaMKIIα-CREB pathway in astrocytes.

Zika virus (ZIKV) infection in the human central nervous system (CNS) causes Guillain-Barre syndrome, cerebellum deformity, and other diseases. Astrocytes are immune response cells in the CNS and an important component of the blood-brain barrier. Consequently, any damage to astrocytes facilitates the spread of ZIKV in the CNS. Connective tissue growth factor/Nephroblastoma overexpressed gene family 1 (CCN1), an important inflammatory factor secreted by astrocytes, is reported to regulate innate immunity and viral infection. However, the mechanism by which astrocyte viral infection affects CCN1 expression remains undefined. In this study, we demonstrate that ZIKV infection up-regulates CCN1 expression in astrocytes, thus promoting intracellular viral replication. Other studies revealed that the cAMP response element (CRE) in the CCN1 promoter is activated by the ZIKV NS3 protein. The cAMP-responsive element-binding protein (CREB), a transacting factor of the CRE, is also activated by NS3 or ZIKV. Furthermore,a specific inhibitor of CREB, i.e. SGC-CBP30, reduced ZIKV-induced CCN1 up-regulation and ZIKV replication. Moreover, co-immunoprecipitation, overexpression, and knockdown studies confirmed that the interaction between NS3 and the regulatory domain of CaMKIIα could activate the CREB pathway, thus resulting in the up-regulation of CCN1 expression and enhancement of virus replication. In conclusion, the findings of our investigations on the NS3-CaMKIIα-CREB-CCN1 pathway provide a foundation for understanding the infection mechanism of ZIKV in the CNS.

ZIKV infection activates the IRE1-XBP1 and ATF6 pathways of unfolded protein response in neural cells.

BACKGROUND: Many viruses depend on the extensive membranous network of the endoplasmic reticulum (ER) for their translation, replication, and packaging. Certain membrane modifications of the ER can be a trigger for ER stress, as well as the accumulation of viral protein in the ER by viral infection. Then, unfolded protein response (UPR) is activated to alleviate the stress. Zika virus (ZIKV) is a mosquito-borne flavivirus and its infection causes microcephaly in newborns and serious neurological complications in adults. Here, we investigated ER stress and the regulating model of UPR in ZIKV-infected neural cells in vitro and in vivo. METHODS: Mice deficient in type I and II IFN receptors were infected with ZIKV via intraperitoneal injection and the nervous tissues of the mice were assayed at 5 days post-infection. The expression of phospho-IRE1, XBP1, and ATF6 which were the key markers of ER stress were analyzed by immunohistochemistry assay in vivo. Additionally, the nuclear localization of XBP1s and ATF6n were analyzed by immunohistofluorescence. Furthermore, two representative neural cells, neuroblastoma cell line (SK-N-SH) and astrocytoma cell line (CCF-STTG1), were selected to verify the ER stress in vitro. The expression of BIP, phospho-elF2α, phospho-IRE1, and ATF6 were analyzed through western blot and the nuclear localization of XBP1s was performed by confocal immunofluorescence microscopy. RT-qPCR was also used to quantify the mRNA level of the UPR downstream genes in vitro and in vivo. RESULTS: ZIKV infection significantly upregulated the expression of ER stress markers in vitro and in vivo. Phospho-IRE1 and XBP1 expression significantly increased in the cerebellum and mesocephalon, while ATF6 expression significantly increased in the mesocephalon. ATF6n and XBP1s were translocated into the cell nucleus. The levels of BIP, ATF6, phospho-elf2α, and spliced xbp1 also significantly increased in vitro. Furthermore, the downstream genes of UPR were detected to investigate the regulating model of the UPR during ZIKV infection in vitro and in vivo. The transcriptional levels of atf4, gadd34, chop, and edem-1 in vivo and that of gadd34 and chop in vitro significantly increased. CONCLUSION: Findings in this study demonstrated that ZIKV infection activates ER stress in neural cells. The results offer clues to further study the mechanism of neuropathogenesis caused by ZIKV infection.

Zika Virus Attenuation by Codon Pair Deoptimization Induces Sterilizing Immunity in Mouse Models.

Zika virus (ZIKV) infection during the large epidemics in the Americas is related to congenital abnormities or fetal demise. To date, there is no vaccine, antiviral drug, or other modality available to prevent or treat Zika virus infection. Here we designed novel live attenuated ZIKV vaccine candidates using a codon pair deoptimization strategy. Three codon pair-deoptimized ZIKVs (Min E, Min NS1, and Min E+NS1) were de novo synthesized and recovered by reverse genetics and contained large amounts of underrepresented codon pairs in the E gene and/or NS1 gene. The amino acid sequence was 100% unchanged. The codon pair-deoptimized variants had decreased replication fitness in Vero cells (Min NS1 ≫ Min E > Min E+NS1), replicated more efficiently in insect cells than in mammalian cells, and demonstrated diminished virulence in a mouse model. In particular, Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer, and a single immunization achieved complete protection against lethal challenge and vertical ZIKV transmission during pregnancy. More importantly, due to the numerous synonymous substitutions in the codon pair-deoptimized strains, reversion to wild-type virulence through gradual nucleotide sequence mutations is unlikely. Our results collectively demonstrate that ZIKV can be effectively attenuated by codon pair deoptimization, highlighting the potential of Min E+NS1 as a safe vaccine candidate to prevent ZIKV infections.IMPORTANCE Due to unprecedented epidemics of Zika virus (ZIKV) across the Americas and the unexpected clinical symptoms, including Guillain-Barré syndrome, microcephaly, and other birth defects in humans, there is an urgent need for ZIKV vaccine development. Here we provided the first attenuated versions of ZIKV with two important genes (E and/or NS1) that were subjected to codon pair deoptimization. Compared to parental ZIKV, the codon pair-deoptimized ZIKVs were mammal attenuated and preferred insect to mammalian cells. Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer and achieved complete protection against lethal challenge and vertical virus transmission during pregnancy. More importantly, the massive synonymous mutational approach made it impossible for the variant to revert to wild-type virulence. Our results have proven the feasibility of codon pair deoptimization as a strategy to develop live attenuated vaccine candidates against flaviviruses such as ZIKV, Japanese encephalitis virus, and West Nile virus.

An improved method for identifying SUMOylation sites of viral proteins.

In this study, we improved the most commonly used methods for MS detection of SUMOylated sites and used an E. coli recombination SUMOylation system with SUMO-1 (T95R). This system provides fast enrichment of SUMOylated viral protein in less than 2 days, and shows advantage over the method of collecting modified protein from cells in convenience and sensitivity. Furthermore, this method provides an option for rapid and accurate identification of the potential viral protein SUMOylation sites.