Two Novel Motifs of Watermelon Silver Mottle Virus NSs Protein Are Responsible for RNA Silencing Suppression and Pathogenicity

The NSs protein of Watermelon silver mottle virus (WSMoV) is the RNA silencing suppressor and pathogenicity determinant. In this study, serial deletion and point-mutation mutagenesis of conserved regions (CR) of NSs protein were performed, and the silencing suppression function was analyzed through agroinfiltration in Nicotiana benthamiana plants. We found two amino acid (aa) residues, H113 and Y398, are novel functional residues for RNA silencing suppression. Our further analyses demonstrated that H113 at the common epitope (CE) (¹⁰⁹KFTMHNQ¹¹⁷), which is highly conserved in Asia type tospoviruses, and the benzene ring of Y398 at the C-terminal β-sheet motif (³⁹⁷IYFL⁴⁰⁰) affect NSs mRNA stability and protein stability, respectively, and are thus critical for NSs RNA silencing suppression. Additionally, protein expression of other six deleted (ΔCR1-ΔCR6) and five point-mutated (Y15A, Y27A, G180A, R181A and R212A) mutants were hampered and their silencing suppression ability was abolished. The accumulation of the mutant mRNAs and proteins, except Y398A, could be rescued or enhanced by co-infiltration with potyviral suppressor HC-Pro. When assayed with the attenuated Zucchini yellow mosaic virus vector in squash plants, the recombinants carrying individual seven point-mutated NSs proteins displayed symptoms much milder than the recombinant carrying the wild type NSs protein, suggesting that these aa residues also affect viral pathogenicity by suppressing the host silencing mechanism.     

Genetic Determinants Responsible for Acquisition of Dengue Type 2 Virus Mouse Neurovirulence

Studies conducted some 50 years ago showed that serial intracerebral passage of dengue viruses in mice selected for neurovirulent mutants that also exhibited significant attenuation for humans. We investigated the genetic basis of mouse neurovirulence of dengue virus because it might be directly or indirectly associated with attenuation for humans. Analysis of the sequence in the C-PreM-E-NS1 region of the parental dengue type 2 virus (DEN2) New Guinea C (NGC) strain and its mouse-adapted, neurovirulent mutant revealed that 10 nucleotide changes occurred during serial passage in mice. Seven of these changes resulted in amino acid substitutions, i.e., Leu₅₅-Phe and Arg₅₇-Lys in PreM, Glu₇₁-Asp, Glu₁₂₆-Lys, Phe₄₀₂-Ile, and Thr₄₅₄-Ile in E, and Arg₁₀₅-Gln in NS1. The sequence of C was fully conserved between the parental and mutant DEN2. We constructed intertypic chimeric dengue viruses that contained the PreM-E genes or only the NS1 gene of neurovirulent DEN2 NGC substituting for the corresponding genes of DEN4. The DEN2 (PreM-E)/DEN4 chimera was neurovirulent for mice, whereas DEN2 (NS1)/DEN4 was not. The mutations present in the neurovirulent DEN2 PreM-E genes were then substituted singly or in combination into the sequence of the nonneurovirulent, parental DEN2. Intracerebral titration of the various mutant chimeras so produced identified two amino acid changes, namely, Glu₇₁-Asp and Glu₁₂₆-Lys, in DEN2 E as being responsible for mouse neurovirulence. The conservative amino acid change of Glu₇₁-Asp probably had a minor effect, if any. The Glu₁₂₆-Lys substitution in DEN2 E, representing a change from a negatively charged amino acid to a positively charged amino acid, most likely plays an important role in conferring mouse neurovirulence.     

Mutagenesis of the NS3 Protease of Dengue Virus Type 2

The flavivirus protease is composed of two viral proteins, NS2B and NS3. The amino-terminal portion of NS3 contains sequence and structural motifs characteristic of bacterial and cellular trypsin-like proteases. We have undertaken a mutational analysis of the region of NS3 which contains the catalytic serine, five putative substrate binding residues, and several residues that are highly conserved among flavivirus proteases and among all serine proteases. In all, 46 single-amino-acid substitutions were created in a cloned NS2B-NS3 cDNA fragment of dengue virus type 2, and the effect of each mutation on the extent of self-cleavage of the NS2B-NS3 precursor at the NS2B-NS3 junction was assayed in vivo. Twelve mutations almost completely or completely inhibited protease activity, 9 significantly reduced it, 14 decreased cleavage, and 11 yielded wild-type levels of activity. Substitution of alanine at ultraconserved residues abolished NS3 protease activity. Cleavage was also inhibited by substituting some residues that are conserved among flavivirus NS3 proteins. Two (Y150 and G153) of the five putative substrate binding residues could not be replaced by alanine, and only Y150 and N152 could be replaced by a conservative change. The two other putative substrate binding residues, D129 and F130, were more freely substitutable. By analogy with the trypsin model, it was proposed that D129 is located at the bottom of the substrate binding pocket so as to directly interact with the basic amino acid at the substrate cleavage site. Interestingly, we found that significant cleavage activity was displayed by mutants in which D129 was replaced by E, S, or A and that low but detectable protease activity was exhibited by mutants in which D129 was replaced by K, R, or L. Contrary to the proposed model, these results indicate that D129 is not a major determinant of substrate binding and that its interaction with the substrate, if it occurs at all, is not essential. This mutagenesis study provided us with an array of mutations that alter the cleavage efficiency of the dengue virus protease. Mutations that decrease protease activity without abolishing it are candidates for introduction into the dengue virus infectious full-length cDNA clone with the aim of creating potentially attenuated virus stocks.     

Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito's RNA Interference Pathway

A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV) infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi), is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA), which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs). These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2) infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti.     

A Meta-Analysis of the Diagnostic Accuracy of Two Commercial NS1 Antigen ELISA Tests for Early Dengue Virus Detection

Background

Dengue virus (DENV) NS1 antigen detection is regarded as an early diagnostic marker. Accordingly, several studies have evaluated the performance of tests that utilize NS1 capture, but the results of individual studies may be limited due to the restricted sample size of the patients recruited. Therefore, our objective was to perform a meta-analysis of the diagnostic accuracy of two commercial NS1 ELISAs (Panbio and Platelia).

Methods and Results

Studies of interest were found in PubMed, Embase and Google Scholar databases using defined inclusion/exclusion criteria. A total of 30 studies containing 12,105 total enrolled patients were included. The results were as follows: 1) Panbio assays showed low overall performance, sensitivity 66% (95% confidence interval (CI) 61–71), specificity 99% (95% CI 96–100), positive likelihood ratio (LR+) 98 (95% CI 20–464), negative likelihood ratio (LR-) 0.3 (95% CI 0.2–0.4), diagnostic odds ratio (DOR) 289 (95% CI 59–1412); 2) Platelia assays showed high overall performance, sensitivity 74% (95% CI 63–82), specificity 99% (95% CI 97–100), LR+ 175 (95% CI 28–1099), LR- 0.3 (95% CI 0.2–0.4), DOR 663 (95% CI 98–4478). The lowest sensitivity values were for secondary infections (57% [95% CI 47–67] and 66% [95% CI 53–77] for Panbio and Platelia, respectively) and for the detection of DENV4. Regarding clinical manifestations, the sensitivity of Platelia was 69% (95% CI 43–86) and 60% (95% CI 48–70) for fever and dengue hemorrhagic fever, respectively. In addition, the sensitivity of both tests was slightly lower for samples from Southeast Asia and Oceania.

Conclusion

DENV1 samples gave higher sensitivity results for both tests. We observed that factors negatively influencing the tests, such as the type of infection, geographical origins of samples and viral serotypes, require further investigation to optimize the diagnostic accuracy.     

CRM1-mediated Nuclear Export of Dengue Virus RNA Polymerase NS5 Modulates Interleukin-8 Induction and Virus Production

Although all established functions of dengue virus NS5 (nonstructural protein 5) occur in the cytoplasm, its nuclear localization, mediated by dual nuclear localization sequences, is essential for virus replication. Here, we have determined the mechanism by which NS5 can localize in the cytoplasm to perform its role in replication, establishing for the first time that it is able to be exported from the nucleus by the exportin CRM1 and hence can shuttle between the nucleus and cytoplasm. We define the nuclear export sequence responsible to be residues 327–343 and confirm interaction of NS5 and CRM1 by pulldown assay. Significantly, greater nuclear accumulation of NS5 during infection due to CRM1 inhibition coincided with altered kinetics of virus production and decreased induction of the antiviral chemokine interleukin-8. This is the first report of a nuclear export sequence within NS5 for any member of the Flavivirus genus; because of its high conservation within the genus, it may represent a target for the treatment of diseases caused by several medically important flaviviruses.The four serotypes of dengue virus (DENV-1–4)2 are the causative agents of the most common arthropod-borne viral disease, dengue fever, and its more severe and potentially deadly dengue hemorrhagic fever form (1). DENV is a member of the genus Flavivirus within the family Flaviviridae. Like all flaviviruses, DENV possesses an ∼11-kb, positive-sense, single-stranded RNA genome that is translated as one long polyprotein and cleaved into 10 viral proteins: three structural (capsid, pre-membrane/membrane, and envelope) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins (2). Flavivirus replication takes place in the cytoplasm, whereby several viral NS and host proteins are believed to constitute the replication complex, the proposed replication machinery of flaviviruses (3). Two key enzymes in replication, NS3 and NS5, the RNA helicase and RNA-dependent RNA polymerase, respectively, interact within the cytoplasm of infected cells (4).The multifunctional NS5 protein is the largest (900 amino acids, 105 kDa) and most highly conserved of the dengue NS proteins (5–7). NS5 contains an N-terminal S-adenosylmethyltransferase domain (5) and a C-terminal RNA-dependent RNA polymerase domain (8–10) separated by an “interdomain linker region” (see Fig. 1). Despite all well established functions of NS5 occurring within the cytoplasm (2), NS5 is predominantly nuclear in DENV-2-infected cells (4, 11).Open in a separate windowFIGURE 1.Top, DENV-2 NS5 possesses two domains, the N-terminal methyltransferase (MET) domain and C-terminal RNA-dependent RNA polymerase (RdRp) domain. The interdomain linker region (residues 320–405, containing two adjacent NLSs) is highlighted. bNLS (residues 320–368) and aNLS (residues 369–405) bind Imp-β/NS3 and Imp-α/β, respectively. Bottom, shown are the GFP-NS5 constructs made in this study. Encoded proteins are shown with the N terminus (residues 1–319; white boxes), bNLS (dotted boxes), aNLS (black boxes), and C terminus (residues 406–900; striped boxes) marked. Hydrophobic regions (termed HR1–HR3) identified as putative NESs are highlighted, and the corresponding alanine substitutions (underlined) generated in this study are indicated (mHR1–mHR3).Proteins >45 kDa require a nuclear localization sequence (NLS) for transport into the nucleus (12, 13). NLSs confer interaction with members of the importin (Imp) superfamily of transporters (either an Imp-α/β heterodimer or Imp-β or a homolog thereof), which mediate the translocation of a cargo into the nucleus. Within the nucleus, the cargo-NLS-Imp complex is dissociated through binding of Ran-GTP to Imp-β, releasing the cargo into the nucleoplasm. Analogously, proteins containing nuclear export sequences (NESs) interact with Imp-β homologs termed exportins, which, when complexed with Ran-GTP, mediate translocation out of the nucleus and into the cytoplasm (14–16). The best characterized of these is CRM1 (exportin 1), which typically binds hydrophobic/leucine-rich NESs (17, 18), such as those of the human immunodeficiency virus Rev protein (19) or the protein kinase A inhibitor PKI (20). The antibiotic leptomycin B (LMB), able to bind CRM1 specifically and to prevent CRM1-NES interaction (21–23), has been widely used to demonstrate the involvement of CRM1 in biological processes (24–28).Previously, we showed that DENV-2 NS5 possesses two NLSs within the interdomain region (see Fig. 1) (29). The C-terminal NLS (amino acids 369–405) or “aNLS” is recognized by Imp-α/β with high affinity and is able to target β-galactosidase to the nucleus in either microinjected or mechanically perforated rat hepatoma cells (29). In contrast, the N-terminal NLS (amino acids 320–368) or “bNLS” is able to bind either Imp-β or NS3 directly in a competitive fashion (30, 31).We demonstrated recently that when NS5 nuclear import is impaired by mutation of the viral genome, the virus is no longer viable, indicating that NS5 nuclear import is essential for virus replication (11). This is attributable, at least in part, to the role of nuclear NS5 in inhibiting induction of the antiviral chemokine interleukin-8 (IL-8) during DENV infection; reduced NS5 nuclear accumulation correlates with increased virus production (11, 32). At least one of the roles of nuclear NS5 is thus to reduce the magnitude of the antiviral response in terms of IL-8 induction.The undisputed role of NS5 is in replication in the cytoplasm. Here, we shed light for the first time on how despite its efficient nuclear localization ability, NS5 is able to fulfill this role. We report the ability of NS5 to be exported from the nucleus in a CRM1-dependent fashion in both transfected and DENV-2-infected cells and identify the NES responsible. Importantly, we show that inhibition of CRM1 during DENV-2 infection results in increased nuclear NS5, reduced IL-8 induction, and increased virus production, underlining the importance of NS5 nuclear export to DENV infection. Our results thus establish for the first time the ability of NS5 to shuttle between the nucleus and cytoplasm and its importance to modulation of the host antiviral response and virus replication.     

The Small Molecules AZD0530 and Dasatinib Inhibit Dengue Virus RNA Replication via Fyn Kinase

In this study, we characterized the antiviral mechanism of action of AZD0530 and dasatinib, two pharmacological inhibitors of host kinases, that also inhibit dengue virus (DV) infection. Using Northern blot and reporter replicon assays, we demonstrated that both small molecules inhibit the DV2 infectious cycle at the step of steady-state RNA replication. In order to identify the cellular target of AZD0530 and dasatinib mediating this anti-DV2 activity, we examined the effects of RNA interference (RNAi)-mediated depletion of the major kinases known to be inhibited by these small molecules. We determined that Fyn kinase, a target of both AZD0530 and dasatinib, is involved in DV2 RNA replication and is probably a major mediator of the anti-DV activity of these compounds. Furthermore, serial passaging of DV2 in the presence of dasatinib led to the identification of a mutation in the transmembrane domain 3 of the NS4B protein that overcomes the inhibition of RNA replication by AZD0530, dasatinib, and Fyn RNAi. Although we observed that dasatinib also inhibits DV2 particle assembly and/or secretion, this activity does not appear to be mediated by Src-family kinases. Together, our results suggest that AZD0530 and dasatinib inhibit DV at the step of viral RNA replication and demonstrate a critical role for Fyn kinase in this viral process. The antiviral activity of these compounds in vitro makes them useful pharmacological tools to validate Fyn or other host kinases as anti-DV targets in vivo.     

Hepatitis C Virus RNA Replication and Virus Particle Assembly Require Specific Dimerization of the NS4A Protein Transmembrane Domain

Hepatitis C virus (HCV) NS4A is a single-pass transmembrane (TM) protein essential for viral replication and particle assembly. The sequence of the NS4A TM domain is highly conserved, suggesting that it may be important for protein-protein interactions. To test this hypothesis, we measured the potential dimerization of the NS4A TM domain in a well-characterized two-hybrid TM protein interaction system. The NS4A TM domain exhibited a strong homotypic interaction that was comparable in affinity to glycophorin A, a well-studied human blood group antigen that forms TM homodimers. Several mutations predicted to cluster on a common surface of the NS4A TM helix caused significant reductions in dimerization, suggesting that these residues form an interface for NS4A dimerization. Mutations in the NS4A TM domain were further examined in the JFH-1 genotype 2a replicon system; importantly, all mutations that destabilized NS4A dimers also caused defects in RNA replication and/or virus assembly. Computational modeling of NS4A TM interactions suggests a right-handed dimeric interaction of helices with an interface that is consistent with the mutational effects. Furthermore, defects in NS4A oligomerization and virus particle assembly of two mutants were rescued by NS4A A15S, a TM mutation recently identified through forward genetics as a cell culture-adaptive mutation. Together, these data provide the first example of a functionally important TM dimer interface within an HCV nonstructural protein and reveal a fundamental role of the NS4A TM domain in coordinating HCV RNA replication and virus particle assembly.     

A Conserved Proline between Domains II and III of Hepatitis C Virus NS5A Influences both RNA Replication and Virus Assembly

We previously demonstrated that two closely spaced polyproline motifs, with the consensus sequence Pro-X-X-Pro-X-Lys/Arg, located between residues 343 to 356 of NS5A, mediated interactions with cellular SH3 domains. The N-terminal motif (termed PP2.1) is only conserved in genotype 1 isolates, whereas the C-terminal motif (PP2.2) is conserved throughout all hepatitis C virus (HCV) isolates, although this motif was shown to be dispensable for replication of the genotype 1b subgenomic replicon. In order to investigate the potential role of these motifs in the viral life cycle, we have undertaken a detailed mutagenic analysis of these proline residues in the context of both genotype 1b (FK5.1) or 2a subgenomic replicons and the genotype 2a infectious clone, JFH-1. We show that the PP2.2 motif is dispensable for RNA replication of all subgenomic replicons and, furthermore, is not required for virus production in JFH-1. In contrast, the PP2.1 motif is only required for genotype 1b RNA replication. Mutation of proline 346 within PP2.1 to alanine dramatically attenuated genotype 1b replicon replication in three distinct genetic backgrounds, but the corresponding proline 342 was not required for replication of the JFH-1 subgenomic replicon. However, the P342A mutation resulted in both a delay to virus release and a modest (up to 10-fold) reduction in virus production. These data point to critical roles for these proline residues at multiple stages in the HCV life cycle; however, they also caution against extrapolation of data from culture-adapted replicons to infectious virus.Hepatitis C virus (HCV) is an enveloped RNA virus which is estimated to infect some 123 million individuals (24). In the majority of cases the virus establishes a chronic infection that can ultimately result in liver fibrosis, cirrhosis, or hepatocellular carcinoma. Thus, there is great interest in elucidating the mechanisms of viral replication, with a view to developing new chemotherapeutic agents. Since 1999, use of the subgenomic replicon system has led to significant progress in the understanding of the mechanism of viral RNA replication. It has been demonstrated that the five nonstructural proteins—NS3, NS4A, NS4B, NS5A, and NS5B—are necessary and sufficient to replicate an RNA molecule containing the 5′ and 3′ untranslated regions (UTRs) of the viral genome. However, apart from the RNA-dependent RNA polymerase (NS5B), the precise details of the roles of each of the nonstructural proteins in the process of RNA replication remain undefined. One problem associated with the subgenomic replicon system is the observation that the replicon RNA undergoes culture adaptation in which, as a result of the error-prone nature of the polymerase, mutations that confer enhanced replicative capacity are selected for in culture. Importantly, it has been shown using the chimpanzee model that, once engineered back into an infectious clone of the virus, such mutations may be attenuating in vivo (5). Recently, the HCV field has been revolutionized by the development of a cell culture infectious system based on a genotype 2a clone derived from a patient with fulminant hepatitis: the JFH-1 clone (30). JFH-1 is also unique in that subgenomic replicons derived from this clone are able to replicate efficiently without culture adaptation. This observation, as well as the fact that full-length genomes of JFH-1 are able to coordinate the coupling of RNA replication to packaging and release of infectious virus particles in Huh7 cells, points to fundamental differences between the RNA replication machinery of JFH-1 and that of the genotype 1b culture-adapted replicons.The majority of mutations conferring culture adaptation map to the region coding for the NS5A protein. NS5A is a zinc-binding phosphoprotein that, as well as playing a critical role in RNA replication, also interacts with a plethora of cellular proteins (18). The protein has been demonstrated to consist of three domains separated by low-complexity sequences (LCS) (29). Of particular interest is the observation that within LCS2 (between domains II and III) (Fig. ​(Fig.1a),1a), NS5A contains two closely spaced polyproline motifs that are able to bind to the SH3 domains of Src-family tyrosine kinases (16), as well as other SH3 domain containing proteins (e.g., Grb2 and amphiphysin II/Bin1) (23, 25, 33). These motifs, which we have termed PP2.1 and PP2.2 (Fig. ​(Fig.1a),1a), conform to the consensus SH3 binding motif Pro-X-X-Pro-X-Arg/Lys, where X is any amino acid (21). Interestingly, although the C-terminal motif (PP2.2) is absolutely conserved in all HCV genotypes, we and others have shown that it is dispensable for HCV RNA replication because alanine substitution of three prolines in this motif (shown to abolish SH3 domain interactions [16]) within a culture-adapted subgenomic replicon had either no effect (19) or resulted in only a modest reduction in replicative capacity (23, 33). The N-terminal motif (PP2.1), however, is only conserved in genotype 1 isolates, although within this motif a proline at residue 346 in genotype 1b (342 in genotype 2a) is absolutely conserved throughout all genotypes, which suggests it has an important role in virus replication.Open in a separate windowFIG. 1.Polyproline motifs in NS5A. (a) Schematic of the structure of NS5A showing the endoplasmic reticulum-membrane associating amphipathic helix (gray box) (4), the position of the coordinated zinc ion, and the three domains with interlinking LCS (black boxes) (29). The lower part of this figure shows the amino acid sequence of the region from residues 343 to 356. These correspond to polyprotein residues 2315 to 2328 in the genotype 1b infectious clone J4 (31). Note that in JFH-1 the corresponding residues in the polyprotein are 2311 to 2325 (residues 339 to 352 within the NS5A sequence). The prolines and basic residues of the SH3 binding motifs (Pro-X-X-Pro-X-Arg/Lys) are in boldface. The accession numbers for the six sequences are as follows: 1a infectious clone H77 (AF009606), 1b infectious clone J4 (AF054247), FK5.1 culture-adapted subgenomic replicon (AJ242654) (13), Con1 isolate (AJ238799), 2a infectious clone JFH-1 (AB047639), and 3a isolate (D17763). (b) Schematic of mutants constructed in the present study. The wild-type FK5.1 sequence is on the top line, residues mutated to alanine indicated by A in the subsequent lines, hyphens indicate unchanged residues.To shed more light on the role of these proline residues in the viral life cycle we have undertaken a mutagenic analysis both in the context of genotype 1b or 2a subgenomic replicons and the cell culture infectious JFH-1 clone. Our results point to key roles of these prolines in multiple stages of virus replication but highlight a surprising discrepancy between the requirements in the two systems.     

抗登革病毒NS1单抗的制备及检测NS1方法的建立

制备抗登革病毒NS1蛋白单克隆抗体,建立检测NS1的ELISA方法。表达1~4型登革病毒NS1蛋白,将1型NS1蛋白纯化后免疫BALB/c小鼠,通过杂交瘤技术制备单克隆抗体。经ELISA、Western blotting、间接免疫荧光筛选和鉴定单克隆抗体,进行纯化和HRP标记。通过鉴定每两株单抗之间是否存在竞争作用,选择非竞争单抗组合并建立NS1捕获法ELISA。结果获得7株高滴度抗NS1单抗,捕获法ELISA可以检出10ng/mL NS1。原核表达登革病毒NS1蛋白制备的单抗可以和天然病毒抗原反应,NS1捕获法ELISA可以用于登革病毒感染检测。     

Two Distant Catalytic Sites Are Responsible for C2c2 RNase Activities

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登革病毒NS3全基因的扩增与克隆

登革热(DF)、登革出血热及登革休克综合征(DHF/DSS)是由登革病毒所致的两种不同临床类型的急性传染病,广泛流行于全球热带及亚热带地区.DHF/DSS以高热、出血、休克、高病死率为主要特征,近年来其发病率有迅速增加的趋势,已成为严重影响人类健康的公共卫生问题.     

登革病毒NS3全基因的扩增与克隆

登革热(ＤＦ)、登革出血热及登革休克综合征(ＤＨＦ/ＤＳＳ)是由登革病毒所致的两种不同临床类型的急性传染病,广泛流行于全球热带及亚热带地区。ＤＨＦ/ＤＳＳ以高热、出血、休克、高病死率为主要特征,近年来其发病率有迅速增加的趋势,已成为严重影响人类健康的公共卫生问题。迄今,ＤＨＦ/ＤＳＳ的发病机制仍不清楚,亦无有效的特异性预防方法[1]。登革病毒属于黄病毒科的黄病毒属,有Ⅰ、Ⅱ、Ⅲ、Ⅳ四个血清型,基因组为单股正链ＲＮＡ,全长约11ｋｂ,编码三种结构蛋白和七种非结构蛋白。基因组顺序为5′ＣＰｒＭＥＮＳ1ＮＳ2ａＮＳ2ｂＮＳ3Ｎ…     

Identification of RNA Binding Proteins Associated with Dengue Virus RNA in Infected Cells Reveals Temporally Distinct Host Factor Requirements

BackgroundThere are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. A better understanding of the host pathogen interaction is required to develop effective therapies to treat DENV. In particular, very little is known about how cellular RNA binding proteins interact with viral RNAs. RNAs within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses) replication.Conclusions/SignificanceThe method for identification of host factors described here is robust and broadly applicable to all RNA viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral RNA within cells. This study significantly increases the number of cellular factors known to interact with DENV and reveals how DENV modulates and usurps cellular proteins for efficient amplification.