Breast milk transmission and involvement of mammary glands in tick-borne flavivirus infected mice.

Tick-borne flaviviruses (TBFs) are transmitted to humans through milk and tick bites. Although a case of possible mother-to-child transmission of tick-borne encephalitis virus (TBEV) through breast milk has been reported, this route has not been confirmed in experimental models. Therefore, in this study, using type I interferon receptor-deficient A129 mice infected with Langat virus (LGTV), we aimed to demonstrate the presence of infectious virus in the milk and mammary glands of infected mice. Our results showed viral RNA of LGTV in the pup's stomach milk clots (SMCs) and blood, indicating that the virus can be transmitted from dam to pup through breast milk. In addition, we observed that LGTV infection causes tissue lesions in the mammary gland, and viral particles were present in mammary gland epithelial cells. Furthermore, we found that milk from infected mice could infect adult mice via the intragastric route, which has a milder infection process, longer infection time, and a lower rate of weight loss than other modes of infection. Specifically, we developed a nano-luciferase-LGTV reporter virus system to monitor the dynamics of different infection routes and observed dam-to-pup infection using in vivo bioluminescence imaging. This study provides comprehensive evidence to support breast milk transmission of TBF in mice and has helped provide useful data for studying TBF transmission routes.IMPORTANCETo date, no experimental models have confirmed mother-to-child transmission of tick-borne flavivirus (TBF) through breastfeeding. In this study, we used a mouse model to demonstrate the presence of infectious viruses in mouse breast milk and mammary gland epithelial cells. Our results showed that pups could become infected through the gastrointestinal route by suckling milk, and the infection dynamics could be monitored using a reporter virus system during breastfeeding in vivo. We believe our findings have provided substantial evidence to understand the underlying mechanism of breast milk transmission of TBF in mice, which has important implications for understanding and preventing TBF transmission in humans.

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.

Discovery and characterization of novel jeilongviruses in wild rodents from Hubei, China.

The genus Jeilongvirus comprises non-segmented negative-stranded RNA viruses that are classified within the Paramyxoviridae family by phylogeny. Jeilongviruses are found in various reservoirs, including rodents and bats. Rodents are typical viral reservoirs with diverse spectra and zoonotic potential. Little is currently known about jeilongviruses in rodents from central China. The study utilized high-throughput and Sanger sequencing to obtain jeilongvirus genomes, including those of two novel strains (HBJZ120/CHN/2021 (17,468 nt) and HBJZ157/CHN/2021 (19,143 nt)) and three known viruses (HBXN18/CHN/2021 (19,212 nt), HBJZ10/CHN/2021 (19,700 nt), HBJM106/CHN/2021 (18,871 nt)), which were characterized by genome structure, identity matrix, and phylogenetic analysis. Jeilongviruses were classified into three subclades based on their topology, phylogeny, and hosts. Based on the amino acid sequence identities and phylogenetic analysis of the L protein, HBJZ120/CHN/2021 and HBJZ157/CHN/2021 were found to be strains rather than novel species. Additionally, according to specific polymerase chain reaction screening, the positive percentage of Beilong virus in Hubei was 6.38%, suggesting that Beilong virus, belonging to the Jeilongvirus genus, is likely to be widespread in wild rodents. The identification of novel strains further elucidated the genomic diversity of jeilongviruses. Additionally, the prevalence of jeilongviruses in Hubei, China, was profiled, establishing a foundation for the surveillance and early warning of emerging paramyxoviruses.

N4-acetylcytidine regulates the replication and pathogenicity of enterovirus 71.

Chemical modifications are important for RNA function and metabolism. N4-acetylcytidine (ac4C) is critical for the translation and stability of mRNA. Although ac4C is found in RNA viruses, the detailed mechanisms through which ac4C affects viral replication are unclear. Here, we reported that the 5' untranslated region of the enterovirus 71 (EV71) genome was ac4C modified by the host acetyltransferase NAT10. Inhibition of NAT10 and mutation of the ac4C sites within the internal ribosomal entry site (IRES) suppressed EV71 replication. ac4C enhanced viral RNA translation via selective recruitment of PCBP2 to the IRES and boosted RNA stability. Additionally, ac4C increased the binding of RNA-dependent RNA polymerase (3D) to viral RNA. Notably, ac4C-deficient mutant EV71 showed reduced pathogenicity in vivo. Our findings highlighted the essential role of ac4C in EV71 infection and provided insights into potential antiviral treatments.

CRISPR/Cas13a Trans-Cleavage-Triggered Catalytic Hairpin Assembly Assay for Specific and Ultrasensitive SARS-CoV-2 RNA Detection.

New coronavirus (SARS-CoV-2), which has caused the coronavirus disease 2019 (COVID-19) pandemic, has brought about a huge burden on global healthcare systems. Rapid and early detection is important to prevent the spread of the pandemic. Here, an assay based on CRISPR/Cas13a and catalytic hairpin assembly (CHA), termed as Cas-CHA, was developed for ultrasensitive and specific detection of SARS-CoV-2 RNA. Upon specific recognition of the target, the CRISPR/Cas13a collaterally cleaved a well-designed hairpin reporter and triggered the CHA reaction. Under optimized conditions, the assay detected the SARS-CoV-2 RNA with a wide range of 100 aM to 100 nM and realized a low detection limit of 84 aM. At the same time, the whole detecting process could be completed within 35 min. More importantly, the assay was able to distinguish SARS-CoV-2 RNA from common human coronaviruses and analyze in saliva samples. By the flexible design of crRNA, the assay was expanded to detect other viruses. The clinical sample analysis verified that the proposed assay held a great potential for practical testing.

Propensity score-matched analysis comparing hippocampus-avoidance whole-brain radiotherapy plus simultaneous integrated boost with hippocampus­avoidance whole-brain radiotherapy alone for multiple brain metastases-a retrospective study in multiple institutions.

BACKGROUND: The optimal treatment for multiple brain metastases has been recently controversially discussed.This study was aimed to explore the feasibility of Hippocampus-Avoidance Whole-Brain Radiotherapy plus a simultaneous integrated boost (HA-WBRT + SIB) in patients with multiple brain metastases and assess tumor control in comparison with Hippocampus-Avoidance Whole-Brain Radiotherapy (HA-WBRT) alone for brain metastases. METHODS: In this study, 63 patients with multiple brain metastases (≥ 4 metastases) had undergone HA-WBRT + SIB between January 2016 and December 2020 in the observation group:HA-WBRT (30 Gy in 12 fractions, the maximum dose of the hippocampus ≤ 14 Gy) plus a simultaneous integrated boost (48 Gy in 12 fractions) for brain metastases.Overall Survival (OS), Median survival,intracranial control (IC = control within the entire brain), intracranial progression-free survival (iPFS) and adverse events were compared with the control group (a HA-WBRT retrospective cohort) by propensity score matching analysis. RESULTS: After 1:1 propensity score matching,there were 56 patients in each group (the observation group, the control group). OS, median survival and iPFS were significantly longer in the observation group (18.4 vs. 10.9 months, P<0.001), (13.0 vs. 8.0 months, P<0.001), (13.9 vs.7.8 months, P<0.001). In comparison of 1-year-IC rates, the observation group also demonstrated higher than the control group (51.8% vs. 21.4%, P = 0.002), respectively. Seven hippocampal metastases were found in the control group (4/56,7.1%) and the observation group (3/56,5.4%) after HA-WBRT. The death rate of intracranial progression were 23.2% in the observation group and 37.5% in the control group.All adverse events were not significant difference between the two groups (P>0.05). CONCLUSIONS: HA-WBRT + SIB resulted in better OS,median survival, IC, iPFS, an acceptable risk of radiation response, and a potential way of declining neurocognitive adverse events, which may be a better treatment for patients with multiple brain metastases.

Crystal structure of a tick-borne flavivirus RNA-dependent RNA polymerase suggests a host adaptation hotspot in RNA viruses.

The RNA-dependent RNA polymerases (RdRPs) encoded by RNA viruses represent a unique class of nucleic acid polymerases. RdRPs are essential in virus life cycle due to their central role in viral genome replication/transcription processes. However, their contribution in host adaption has not been well documented. By solving the RdRP crystal structure of the tick-borne encephalitis virus (TBEV), a tick-borne flavivirus, and comparing the structural and sequence features with mosquito-borne flavivirus RdRPs, we found that a region between RdRP catalytic motifs B and C, namely region B-C, clearly bears host-related diversity. Inter-virus substitutions of region B-C sequence were designed in both TBEV and mosquito-borne Japanese encephalitis virus backbones. While region B-C substitutions only had little or moderate effect on RdRP catalytic activities, virus proliferation was not supported by these substitutions in both virus systems. Importantly, a TBEV replicon-derived viral RNA replication was significantly reduced but not abolished by the substitution, suggesting the involvement of region B-C in viral and/or host processes beyond RdRP catalysis. A systematic structural analysis of region B-C in viral RdRPs further emphasizes its high level of structure and length diversity, providing a basis to further refine its relevance in RNA virus-host interactions in a general context.

The influence mechanism of community-built environment on the health of older adults: from the perspective of low-income groups.

BACKGROUND: With the rapid development of population ageing, the international community has been paying more attention to the health problems of older adults and the age-friendly community. But there has not been enough discussion about the internal mechanism of the community-built environment that influences the health of older adults. The aim of our study was to explore the complex relationships among community-built environment, social participation, outdoor exercise, and health of older adults, as well as the differences among older adults in different income groups, particular attention was paid to the situation of low-income group. METHODS: This study used descriptive statistical analysis and structural equation Modeling (SEM) to make a group comparison among older adults in different income groups. The data of this study came from a sample survey in Shanghai, China. RESULTS: The study found that health difference exists among older adults in China: the lower the income, the worse the community-built environment, the worse the health. The community-built environment had an important impact on the health of older adults, especially the low-income older adults. And the community-built environment influenced the health of older adults through the intermediary role of outdoor exercise and social participation. Furthermore, the lower the income level of older adults, the stronger the direct effect of the community-built environment on their health; the higher the income level of older adults, the stronger the mediating effect of outdoor exercise and social participation on the impact of the community-built environment on their health. CONCLUSION: Governments should pay more attention to the health and living conditions of low-income older adults and take proactive steps to help them. Community design and construction should pay more attention to the demands of low-income older adult groups, which will help to improve the health inequality of older adults, consequently enhancing older adults' overall health.

DNAzyme Walker for Homogeneous Detection of Enterovirus EV71 and CVB3.

When dealing with infectious pathogens, the risk of contamination or infection in the process of detecting them is nonnegligible. Separation-free detection will be beneficial in operation and safety. In this work, we proposed a DNAzyme walker for homogeneous and isothermal detection of enterovirus. The DNAzyme is divided into two inactivate subunits. When the subunit-conjugated antibody binds to the target virus, the activity of the DNAzyme recovers as a result of spatial proximity. The walker propels, and the fluorescence recovers. The final fluorescence intensity of the reaction mixture is related to the concentration of the target virus. The detection limit of this proposed method is 6.6 × 104 copies/mL for EV71 and 4.3 × 104 copies/mL for CVB3, respectively. Besides, this method was applied in detection of EV71 in clinical samples with a satisfactory result. The entire experiment is easy to operate, and the proposed method has great potential for practical use.

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.

One-Step Monitoring of Multiple Enterovirus 71 Infection-Related MicroRNAs Using Core-Satellite Structure of Magnetic Nanobeads and Multicolor Quantum Dots.

The accurate and rapid monitoring of the expression levels of enterovirus 71 (EV71)-related microRNAs (miRNAs) can contribute to diagnosis of hand, foot, and mouth disease (HFMD) at the early stage. However, there is currently a lack of convenient methods for simultaneous monitoring of multiplex miRNAs in one step. Herein a one-step method for the simultaneous monitoring of multiple EV71 infection-related miRNAs is developed based on core-satellite structure assembled with magnetic nanobeads and quantum dots (MNs-ssDNA-QDs). In the presence of target miRNAs, duplex-specific nuclease (DSN)-assisted target recycling can be triggered, resulting in the release of QDs and recycling of target miRNAs. Then the simultaneous quantification can be easily realized by recording the corresponding amplified fluorescence signal of QDs in the suspension. With this method, simultaneous detection of hsa-miRNA-296-5p and hsa-miRNA-16-5p, potential biomarkers of EV71 infection, can be easily achieved with femtomolar sensitivity and single-base mismatch specificity. Moreover, the method is successfully used for monitoring of the expression level of miRNAs in EV71-infected cells at different time points, demonstrating the potential for diagnostic applications. With the merits of one-step operation and single-nucleotide mismatch discrimination, this work opens a new avenue for multiplex miRNAs detection. As different nucleotide sequences and multicolor QDs can be employed, this work is expected to offer great potential for the development of high throughput diagnosis.

Dissecting the Cell Entry Pathway of Baculovirus by Single-Particle Tracking and Quantitative Electron Microscopic Analysis.

The budded virus of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infects insect cells through mainly clathrin-mediated endocytosis. However, the cell entry pathway of AcMNPV remains unclear. In this study, by using population-based analysis of single-virus tracking and electron microscopy, we investigated the internalization, fusion behavior, and endocytic trafficking of AcMNPV. AcMNPV internalization into host insect cells was facilitated by actin polymerization and dynamin. After incorporation into early endosomes, the AcMNPV envelope fused with the membranes of early endosome, allowing for nucleocapsid release into the cytoplasm. Microtubules were implicated in the bidirectional and long-range transport of virus-containing endosomes. In addition, microtubule depolymerization reduced the motility of virus-bearing early endosomes, impairing the progression of infection beyond enlarged early endosomes. These findings demonstrated that AcMNPV internalization was facilitated by actin polymerization in a dynamin-dependent manner, and nucleocapsid release occurred in early endosomes in a microtubule-dependent manner. This study provides mechanistic and kinetic insights into AcMNPV infection and enhance our understanding of the infection pathway of baculoviruses.IMPORTANCE Baculoviruses are used widely as environmentally benign pesticides, protein expression systems, and potential mammalian gene delivery vectors. Despite the significant application value, little is known about the cell entry and endocytic trafficking pathways of baculoviruses. In this study, we demonstrated that the alphabaculovirus AcMNPV exhibited actin- and microtubule-dependent transport for nucleocapsid release predominantly from within early endosomes. In contrast to AcMNPV transduction in mammalian cells, its infection in host insect cells is facilitated by actin polymerization for internalization and microtubules for endocytic trafficking within early endosomes, implying that AcMNPV exhibits cell type specificity in the requirement of the cytoskeleton network. In addition, experimental depolymerization of microtubules impaired the progression of infection beyond enlarged early endosomes. This is the first study that dissects the cell entry pathway of baculoviruses in host cells at the single-particle level, which advances our understanding of the early steps of baculovirus entry.

Age sequences of the elderly' social network and its efficacies on well-being: an urban-rural comparison in China.

BACKGROUND: Although social network is a known determinant of the elderly's well-being, it is not clear, in urban-rural and age-comparison, what its structural characteristics are and how it works for well-being. The research aims to discuss the features of the elderly's social network and the social network efficacies on the well-being of older adults in China's urban and rural areas as well as revealing the urban-rural disparities among the elderly of different age groups. METHODS: In this study, descriptive statistical analysis and structural equation Modeling (SEM) were used to make a group comparison between the urban and rural elderly of different age groups. All data are quoted from 2014 China Longitudinal Aging Social Survey (CLASS). The survey adopted the multi-stage probability sampling method, targeting Chinese senior citizens aged 60 and above, the ultimate samples totaled 11,511. RESULTS: The social network of the elderly in China feature a "reverse structure" in age sequences: with ageing, family network of the elderly expand while their friend network shrink; also, the expansion scale of the rural elderly's family network is significantly larger than that of the city's while the shrinkage scale of their friend network is smaller compared with its urban counterpart. The effect of family network on the rural elderly's well-being shows a remarkable increase with age. However, there is no noticeable change in urban elderly groups of different ages. CONCLUSION: The social network characteristics of the Chinese elderly are different between different age stages. Namely, the family network and the friend network have the "reverse structure " in age sequences. Meanwhile, the family network and the friend network have different efficacies on the well-being of the elderly in China, and the differences between urban and rural areas are even more obvious. For rural elderly, family network has very important effects on their well-being. Moreover, With the increase of age, family network's efficacies increase gradually. For urban elderly, comparatively, family network is just as important as friend network.

Target-Induced Cascade Amplification for Homogeneous Virus Detection.

Detection of viruses with high sensitivity is critical for the prevention and treatment of the related disease. Two homogeneous target-induced cascade amplification methods were proposed for the detection of enterovirus 71 and coxsackievirus B3. These methods both employ DNAzyme but differ in the way in which the DNAzyme is amplified. In the hybridization chain reaction (HCR)-based strategy, the DNAzyme is assembled by hairpin DNA strands, while in the rolling circle amplification (RCA)-based strategy, the DNAzyme is synthesized by the polymerase. On the basis of the virion structure, we investigated the effects of using only VP1-antibody or VP1-antibody and VP2-antibody on the detection. And the combination of two kinds of antibodies was found to further improve the performance of the detection. Subsequently, the simultaneous detection of EV71 and CVB3 was achieved by the RCA-based strategy. And the proposed methods were also applied in clinical samples analysis with a satisfactory result, showing great potential for applications in virus detection.

Multifunctional Cellular Beacons with in Situ Synthesized Quantum Dots Make Pathogen Detectable with the Naked Eye.

The rapid and sensitive detection of pathogens is extremely crucial for timely clinical diagnosis and diseases control. Here, by employing cellular beacons with in situ synthesized QDs created from Staphylococcus aureus ( S. aureus), we efficiently fabricated an antibody (Ab) and acetylcholinesterase (AChE)-functionalized nanobioprobe, i.e., multifunctional cellular beacons (MCBs), avoiding complicated modification. Coupled with magnetic separation, a novel method for pathogen detection with the naked eye is established. With this method, enterovirus 71 (EV71) can be detected by the naked eye through the aggregation of gold nanoparticles that is triggered by the product of AChE catalyzed acetylthiocholine, with a detection limit of 0.5 ng/mL. Moreover, due to the MCBs have high luminance with perfect uniformity, the detection can also be realized by counting the number of MCBs, with a detection limit of 1 ng/mL. The method is validated with human throat swabs, resulting in a complete consistence with reverse transcription-polymerase chain reaction results. This study reports the first cellular beacons-based method for pathogen detection by the naked eye and broadens the applicability of cell self-synthesized nanoparticles-based immunoassays. Moreover, the MCBs-based method will provide a powerful tool for clinical detection.

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.

The 3C protease of enterovirus A71 counteracts the activity of host zinc-finger antiviral protein (ZAP).

Enterovirus A71 (EV-A71) is a positive-strand RNA virus that causes hand-foot-mouth disease and neurological complications in children and infants. Although the underlying mechanisms remain to be further defined, impaired immunity is thought to play an important role. The host zinc-finger antiviral protein (ZAP), an IFN-stimulated gene product, has been reported to specifically inhibit the replication of certain viruses. However, whether ZAP restricts the infection of enteroviruses remains unknown. Here, we report that EV-A71 infection upregulates ZAP mRNA in RD and HeLa cells. Moreover, ZAP overexpression rendered 293 T cells resistant to EV-A71 infection, whereas siRNA-mediated depletion of endogenous ZAP enhanced EV-A71 infection. The EV-A71 infection stimulated site-specific proteolysis of two ZAP isoforms, leading to the accumulation of a 40 kDa N-terminal ZAP fragment in virus-infected cells. We further revealed that the 3C protease (3Cpro) of EV-A71 mediates ZAP cleavage, which requires protease activity. Furthermore, ZAP variants with single amino acid substitutions at Gln-369 were resistant to 3Cpro cleavage, implying that Gln-369 is the sole cleavage site in ZAP. Moreover, although ZAP overexpression inhibited EV-A71 replication, the cleaved fragments did not show this effect. Our results indicate that an equilibrium between ZAP and enterovirus 3Cpro controls viral infection. The findings in this study suggest that viral 3Cpro mediated ZAP cleavage may represent a mechanism to escape host antiviral responses.

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.

2C Proteins of Enteroviruses Suppress IKKß Phosphorylation by Recruiting Protein Phosphatase 1.

UNLABELLED: The NF-κB signaling network, which is an ancient signaling pathway, plays a pivotal role in innate immunity and constitutes a first line of defense against invading pathogens, including viruses. However, numerous viruses possess evolved strategies to antagonize the activation of the NF-κB signaling pathway. Our previous study demonstrated that the nonstructural protein 2C of enterovirus 71 (EV71), which is the major pathogen of hand, foot, and mouth disease, inhibits tumor necrosis factor alpha (TNF-α)-mediated activation of NF-κB by suppressing IκB kinase ß (IKKß) phosphorylation. Nevertheless, the mechanism underlying the inhibition of IKKß phosphorylation by EV71 2C remains largely elusive. We demonstrate that EV71 2C interacts with all isoforms of the protein phosphatase 1 (PP1) catalytic subunit (the PP1α, PP1ß, and PP1γ isoforms) through PP1-docking motifs. EV71 2C has no influence on the subcellular localization of PP1. In addition, the PP1-binding-deficient EV71 2C mutant 3E3L nearly completely lost the ability to suppress IKKß phosphorylation and NF-κB activation was markedly restored in the mutant, thereby indicating that PP1 binding is efficient for EV71 2C-mediated inhibition of IKKß phosphorylation and NF-κB activation. We further demonstrate that 2C forms a complex with PP1 and IKKß to dephosphorylate IKKß. Notably, we reveal that other human enteroviruses, including poliovirus (PV), coxsackie A virus 16 (CVA16), and coxsackie B virus 3 (CVB3), use 2C proteins to recruit PP1, leading to the inhibition of IKKß phosphorylation. Our findings indicate that enteroviruses exploit a novel mechanism to inhibit IKKß phosphorylation by recruiting PP1 and IKKß to form a complex through 2C proteins, which ultimately results in the inhibition of the NF-κB signaling pathway. IMPORTANCE: The innate antiviral immunity system performs an essential function in recognizing and eliminating invading viruses. Enteroviruses include a number of important human pathogens, including poliovirus (PV), EV71, and coxsackieviruses (CVs). As 2C is the most conserved and complex nonstructural protein of enteroviruses, its biological function is largely unclear, whereas the 2A and 3C proteinases of enteroviruses are well characterized. We reveal that EV71 2C forms a complex with PP1 and IKKß to maintain IKKß in an unphosphorylated and inactive state, resulting in the inactivation of the TNF-α-mediated NF-κB signaling pathway. We provide evidence that the 2C proteins of the enteroviruses PV, CVA16, and CVB3 suppress IKKß phosphorylation through the same mechanism involving PP1. We demonstrate that enteroviruses exploit a novel mechanism involving PP1 to regulate innate antiviral immunity, and our findings may be particularly important for understanding the pathogenicity of enteroviruses.

SUMO Modification Stabilizes Enterovirus 71 Polymerase 3D To Facilitate Viral Replication.

Accumulating evidence suggests that viruses hijack cellular proteins to circumvent the host immune system. Ubiquitination and SUMOylation are extensively studied posttranslational modifications (PTMs) that play critical roles in diverse biological processes. Cross talk between ubiquitination and SUMOylation of both host and viral proteins has been reported to result in distinct functional consequences. Enterovirus 71 (EV71), an RNA virus belonging to the family Picornaviridae, is a common cause of hand, foot, and mouth disease. Little is known concerning how host PTM systems interact with enteroviruses. Here, we demonstrate that the 3D protein, an RNA-dependent RNA polymerase (RdRp) of EV71, is modified by small ubiquitin-like modifier 1 (SUMO-1) both during infection and in vitro Residues K159 and L150/D151/L152 were responsible for 3D SUMOylation as determined by bioinformatics prediction combined with site-directed mutagenesis. Also, primer-dependent polymerase assays indicated that mutation of SUMOylation sites impaired 3D polymerase activity and virus replication. Moreover, 3D is ubiquitinated in a SUMO-dependent manner, and SUMOylation is crucial for 3D stability, which may be due to the interplay between the two PTMs. Importantly, increasing the level of SUMO-1 in EV71-infected cells augmented the SUMOylation and ubiquitination levels of 3D, leading to enhanced replication of EV71. These results together suggested that SUMO and ubiquitin cooperatively regulated EV71 infection, either by SUMO-ubiquitin hybrid chains or by ubiquitin conjugating to the exposed lysine residue through SUMOylation. Our study provides new insight into how a virus utilizes cellular pathways to facilitate its replication. IMPORTANCE: Infection with enterovirus 71 (EV71) often causes neurological diseases in children, and EV71 is responsible for the majority of fatalities. Based on a better understanding of interplay between virus and host cell, antiviral drugs against enteroviruses may be developed. As a dynamic cellular process of posttranslational modification, SUMOylation regulates global cellular protein localization, interaction, stability, and enzymatic activity. However, little is known concerning how SUMOylation directly influences virus replication by targeting viral polymerase. Here, we found that EV71 polymerase 3D was SUMOylated during EV71 infection and in vitro Moreover, the SUMOylation sites were determined, and in vitro polymerase assays indicated that mutations at SUMOylation sites could impair polymerase synthesis. Importantly, 3D is ubiquitinated in a SUMOylation-dependent manner that enhances the stability of the viral polymerase. Our findings indicate that the two modifications likely cooperatively enhance virus replication. Our study may offer a new therapeutic strategy against virus replication.