Xiao-Yan Wu

Human enterovirus 71 uncoating captured at atomic resolution.

ABSTRACT Human enterovirus 71 (EV71) is the major causative agent of severe hand-foot-and-mouth diseases (HFMD) in young children, and structural characterization of EV71 during its life cycle can aid in the development of therapeutics against HFMD. Here, we present the atomic structures of the full virion and an uncoating intermediate of a clinical EV71 C4 strain to illustrate the structural changes in the full virion that lead to the formation of the uncoating intermediate prepared for RNA release. Although the VP1 N-terminal regions observed to penetrate through the junction channel at the quasi-3-fold axis in the uncoating intermediate of coxsackievirus A16 were not observed in the EV71 uncoating intermediate, drastic conformational changes occur in this region, as has been observed in all capsid proteins. Additionally, the RNA genome interacts with the N-terminal extensions of VP1 and residues 32 to 36 of VP3, both of which are situated at the bottom of the junction. These observations highlight the importance of the junction for genome release. Furthermore, EV71 uncoating is associated with apparent rearrangements and expansion around the 2- and 5-fold axes without obvious changes around the 3-fold axes. Therefore, these structures enabled the identification of hot spots for capsid rearrangements, which led to the hypothesis that the protomer interface near the junction and the 2-fold axis permits the opening of large channels for the exit of polypeptides and viral RNA, which is an uncoating mechanism that is likely conserved in enteroviruses. IMPORTANCE Human enterovirus 71 (EV71) is the major causative agent of severe hand-foot-and-mouth diseases (HFMD) in young children. EV71 contains an RNA genome protected by an icosahedral capsid shell. Uncoating is essential in EV71 life cycle, which is characterized by conformational changes in the capsid to facilitate RNA release into host cell. Here we present the atomic structures of the full virion and an uncoating intermediate of a clinical C4 strain of EV71. Structural analysis revealed drastic conformational changes associated with uncoating in all the capsid proteins near the junction at the quasi-3-fold axis and protein-RNA interactions at the bottom of the junction in the uncoating intermediate. Significant capsid rearrangements also occur at the icosahedral 2- and 5-fold axes but not at the 3-fold axis. Taking the results together, we hypothesize that the junction and nearby areas are hot spots for capsid breaches for the exit of polypeptides and viral RNA during uncoating.

Parallel mRNA and microRNA profiling of HEV71-infected human neuroblastoma cells reveal the up-regulation of miR-1246 in association with DLG3 repression.

Human enterovirus 71 (HEV71) has emerged as the leading cause of viral encephalitis in children in most Asian countries. The roles of host miRNAs in the neurological pathogenesis of HEV71 infection remain unknown. In the present study, comprehensive miRNA expression profiling in HEV71-infected human neuroblastoma SH-SY5Y cells was performed using the Affymetrix Gene Chip microarray assay and was validated using real-time RT-PCR. Among the 69 differentially expressed miRNAs, miR-1246 was specifically induced by HEV71 infection in human neuroblastoma cells, but inhibition of miR-1246 failed to affect HEV71 replication. Parallel mRNA and microRNA profiling based on the 35 K Human Genome Array identified 182 differentially regulated genes. Target prediction of miR-1246 and network modeling revealed 14 potential target genes involved in cell death and cell signaling. Finally, a combined analysis of the results from mRNA profiling and miR-1246 target predication led to the identification of disc-large homolog 3 (DLG3), which is associated with neurological disorders, for further validation. Sequence alignment and luciferase reporter assay showed that miR-1246 directly bound with the 3′-UTR of DLG3 gene. Down-regulation of miR-1246 induced significant changes in DLG3 expression levels in HEV71-infected SHSY5Y cells. Together, these results suggested that miR-1246 might play a role in neurological pathogenesis of HEV71 by regulating DLG3 gene in infected cells. These findings provide new information on the miRNA and mRNA profiles of HEV71-infected neuroblastoma cells. The biological significance of miR-1246 and DLG3 during the course of HEV71 infection deserves further investigation.

Global Transcriptomic Analysis of Human Neuroblastoma Cells in Response to Enterovirus Type 71 Infection

Human enterovirus type 71 (EV71) is the major pathogen of hand-foot-and-mouth disease (HFMD) and has been associated with severe neurological disease and even death in infants and young children. The pathogenesis of EV71 infection in the human central nervous system remains unclear. In this study, human whole genome microarray was employed to perform transcriptome profiling in SH-SY5Y human neuroblastoma cells infected with EV71. The results indicated that EV71 infection lead to altered expression of 161 human mRNAs, including 74 up-regulated genes and 87 down-regulated genes. Bioinformatics analysis indicated the possible roles of the differentially regulated mRNAs in selected pathways, including cell cycle/proliferation, apoptosis, and cytokine/chemokine responses. Finally, the microarray results were validated using real-time RT-PCR with high identity. Overall, our results provided fundamental information regarding the host response to EV71 infection in human neuroblastoma cells, and this finding will help explain the pathogenesis of EV71 infection and virus-host interaction.

PB1-mediated virulence attenuation of H5N1 influenza virus in mice is associated with PB2

H5N1 avian influenza viruses demonstrate different phenotypes, such as pathogenicity after one or serial passages in mammalian hosts or cells. To establish the molecular basis of these phenotypes, we cloned isolates from the lungs of mice infected with human A/Vietnam/1194/2004 (H5N1) influenza virus. Large-plaque isolates were less pathogenic to mice than small-plaque isolates. Genome sequencing revealed that the small-plaque and large-plaque isolates differed in several amino acids. In order to assess their effects on pathogenicity in mice, two amino acid changes common to attenuated isolates, one in PB2 (I63T) and the other in PB1 (T677M), were inserted into a wild-type recombinant virus construct. The PB2 (I63T) or PB1 (T677M) mutations alone did not alter the phenotype of H5N1 virus, whereas recombinant virus with both mutations was less pathogenic than the wild-type recombinant virus. Furthermore, the PB1 (T677M) mutation showed a lower replication efficiency, although it had higher polymerase activity. The recombinant virus with the PB2 (63T) mutation replicated as well as the wild-type recombinant virus. These results suggest that the C terminus of PB1 of H5N1 influenza virus mediates virulence attenuation of H5N1 influenza virus in mice, associating with the N terminus of PB2. However, the role of the N terminus of PB2 in virulence attenuation in mice remains unclear.

In vitro characterization of human adenovirus type 55 in comparison with its parental adenoviruses, types 11 and 14.

Human adenovirus type 55 (HAdV-B55) represents a re-emerging human pathogen, and this adenovirus has been reported to cause outbreaks of acute respiratory diseases among military trainees and in school populations around the world. HAdV-B55 has been revealed to have evolved from homologous recombination between human adenovirus type 14 (HAdV-B14) and type 11 (HAdV-B11), but it presents different clinical manifestations from parental virus HAdV-B11. In the present paper, we report the distinct biological features of HAdV-B55 in comparison with the parental viruses HAdV-B11 and HAdV-B14 in cell cultures. The results showed that HAdV-B55 replicated well in various cells, similar to HAdV-B11 and HAdV-B14, but that its processing had a slower and milder cytopathic effect in the early stages of infection. Viral fitness analysis showed that HAdV-B55 exhibited higher levels of replication in respiratory cells than did either of its parents. Cytotoxicity and apoptosis analyses in A549 cells indicated that HAdV-B55 was less cytotoxic than HAdV-B11 and HAdV-B14 were and induced milder apoptosis. Finally, thermal sensitivity analysis revealed that HAdV-B55 exhibited lower thermostability than did either HAdV-B11 or HAdV-B14, which may limit the transmission of HAdV-B55 in humans. Together, the findings described here expand current knowledge about this re-emerging recombinant HAdV, shedding light on the pathogenesis of HAdV-B55.

Elevation of Matrix Metalloproteinase-9 Level in Cerebrospinal Fluid of Tick-Borne Encephalitis Patients Is Associated with IgG Extravassation and Disease Severity

Background Tick-borne encephalitis (TBE), caused by tick-borne encephalitis virus (TBEV), is an infectious disease involving the central nervous system (CNS). The pathogenesis of CNS injury has not been clearly demonstrated. Matrix metalloproteinase-9 (MMP-9) and some cytokines, such as interleukin 6 (IL-6), may play important roles in the disruption of the blood-brain barrier (BBB) and the pathogenesis of TBE. Methods 72 cerebrospinal fluid (CSF) samples were collected from TBE patients in north eastern China. IgG levels in CSF and serum were compared and MMP-9 and IL-6 levels were evaluated by ELISA. The correlation between the elevated MMP-9 levels and IgG extravasation, disease severity, and neuroinflammation was analyzed. Results Increased concentration of MMP-9 was detected in some of the CSF samples, and the elevation was found to be closely related to CSF TBEV IgG extravasation and enhancement of IL-6 expression. Moreover, elevated levels of MMP-9 were found to be correlated with IL-6 enhancement. Four of the 72 patients, the ones who died, presented with high CSF MMP-9 levels. Conclusions In TBE patients, elevated CSF MMP-9 levels were associated with brain inflammatory reaction, disruption of the blood-brain barrier, and disease severity.

Human enterovirus co-infection in severe HFMD patients in China.

specimens Virus isolation in different cell lines Clinical manifestation MEK RD Vero SD2012-2 EV71 CVB3 severe SD2012-4 EV71 CVB3 severe SD2012-5 EV71 CVB3 severe SD2012-7 CVA10 CVB3 severe SD2012-8 CVA16 CVA10 mild SD2012-12 CVA10 CVB3 mild mild CVA10 SD2012-23 mild CVA16 SD2012-25 mild CVB3 SD2012-28 mild EV71 SD2012-37 severe EV71 EV71 SD2012-42 severe EV71 EV71 SD2012-43 severe EV71 EV71 SD2012-55 ... SD2012-99 ... EV71 ... ... ... severe

U4 at the 3′ UTR of PB1 Segment of H5N1 Influenza Virus Promotes RNA Polymerase Activity and Contributes to Viral Pathogenicity

The viral RNA-dependent RNA polymerase has been found to contribute to efficient replication in mammalian systems and to the high pathogenicity of H5N1 influenza A virus in humans and other mammals. The terminal untranslated regions of the viral segments perform functions such as polyadenylation and contain signals for genomic packaging and initiation of RNA synthesis. These sequences are highly conserved, apart from a U/C polymorphism at position 4 of the 3′ end, most often seen in the polymerase gene segments. However, no study has yet tested whether the untranslated regions of H5N1 make any contribution to its high pathogenicity. Herein, the association of the fourth nucleotide at the 3′ end of the untranslated region in segment 2 (PB1), of A/Vietnam/1194/2004 (H5N1), with pathogenicity was examined in mice. To this end, an RNA polymerase reporter system was constructed, and viruses with mutations at this site were rescued. Results showed the U4 in PB1 was found to contribute to greater amounts of RNA-dependent RNA polymerase activity and differentially regulate genomic transcription and replication. Although a recombinant H5N1 virus with the rarer C4 sequence in all eight segments was viable and replicated to high titers in vitro, replacing a single U4 at the 3′ termini of the PB1 gene segment enhanced viral reproduction and more pathogenesis. In this way, these data showed the importance of untranslated regions of H5N1 influenza virus to pathogenicity.

Construction and genetic analysis of murine hepatitis virus strain A59 Nsp16 temperature sensitive mutant and the revertant virus.

Coronaviruses (CoVs) are generally associated with respiratory and enteric infections and have long been recognized as important pathogens of livestock and companion animals. Mouse hepatitis virus (MHV) is a widely studied model system for Coronavirus replication and pathogenesis. In this study, we created a MHV-A59 temperature sensitive (ts) mutant Wu”-ts18(cd) using the recombinant vaccinia reverse genetics system. Virus replication assay in 17C1-1 cells showed the plaque phenotype and replication characterization of constructed Wu”-ts18(cd) were indistinguishable from the reported ts mutant Wu”-ts18. Then we cultured the ts mutant Wu”-ts18(cd) at non-permissive temperature 39.5°C, which “forced” the ts recombinant virus to use second-site mutation to revert from a ts to a non-ts phenotype. Sequence analysis showed most of the revertants had the same single amino acid mutation at Nsp16 position 43. The single amino acid mutation at Nsp16 position 76 or position 130 could also revert the ts mutant Wu”-ts18 (cd) to non-ts phenotype, an additional independent mutation in Nsp13 position 115 played an important role on plaque size. The results provided us with genetic information on the functional determinants of Nsp16. This allowed us to build up a more reasonable model of CoVs replication-transcription complex.

The confirmation of three repeated sequence elements in the 3′ untranslated region of Chikungunya virus

In this study, the complete genomic nucleotide sequence of Chikungunya virus (CHIKV) strain S27 African prototype was determined and three 21 nucleotides repeated sequence elements (RSEs) at positions 11398–11418, 11533–11553, and 11620–11640 in the 3′ untranslated region (3′UTR) were confirmed. In addition, the 3′UTRs of all CHIKV strains deposited in GenBank were analyzed. The results displayed that the majority of the CHIKV strains consisted of the three 21 nucleotides RSEs in the 3′UTRs, and the third RSE was the most conservative. The conservation of the three RSEs of 21 nucleotides within the 3′UTR of CHIKV genome may play an important role on the virus replication cycle.