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.     

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.     

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.     

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.     

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.     

Two Distant Catalytic Sites Are Responsible for C2c2 RNase Activities

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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.     

Flexibility of NS5 Methyltransferase-Polymerase Linker Region Is Essential for Dengue Virus Replication

We examined the function of the conserved Val/Ile residue within the dengue virus NS5 interdomain linker (residues 263 to 272) by site-directed mutagenesis. Gly substitution or Gly/Pro insertion after the conserved residue increased the linker flexibility and created slightly attenuated viruses. In contrast, Pro substitution abolished virus replication by imposing rigidity in the linker and restricting NS5''s conformational plasticity. Our biochemical and reverse genetics experiments demonstrate that NS5 utilizes conformational regulation to achieve optimum viral replication.     

Localization and Functions of Japanese Encephalitis Virus Nonstructural Proteins NS3 and NS5 for Viral RNA Synthesis in the Infected Cells

Recently it has been reported that Japanese encephalitis virus (JEV)-specific RNAs can be synthesized in vitro in the subcellular fraction including outer-nuclear membrane (Takegami and Hotta, 1989). The results of Western blot analysis and indirect immunofluorescence test using two kinds of monospecific antisera against JEV nonstructural proteins NS3 and NS5 showed that NS3 and NS5 were membrane-associated proteins and formed the complex at the perinuclear site in the infected cells. Both antisera against NS3 and NS5 inhibited in vitro RNA synthesis. These results suggest that NS5 and NS3 play important role(s) in flavivirus RNA replication.     

Distinct Structural Features of Cyclothiazide Are Responsible for Effects on Peak Current Amplitude and Desensitization Kinetics at iGluR2

Ionotropic glutamate receptors (iGluRs) mediate fast excitatory neurotransmission. Upon glutamate application, 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid receptors undergo rapid and almost complete desensitization that can be attenuated by positive allosteric modulators. The molecular mechanism of positive allosteric modulation has been elucidated previously by crystal structures of the ligand-binding core of iGluR2 in complex with, for example, cyclothiazide (CTZ). Here, we investigate the structure and function of CTZ and three closely related analogues NS1493, NS5206, and NS5217 at iGluR2, by X-ray crystallography and fast application patch-clamp electrophysiology. CTZ was the most efficacious and potent modulator of the four compounds on iGluR2(Q)_i [E_max normalized to response of glutamate: 754% (CTZ), 490% (NS1493), 399% (NS5206), and 476% (NS5217) and EC₅₀ in micromolar: 10 (CTZ), 26 (NS1493), 43 (NS5206), and 48 (NS5217)]. The four modulators divide into three groups according to efficacy and desensitization kinetics: (1) CTZ increases the peak current efficacy twice as much as the three analogues and nearly completely blocks receptor desensitization; (2) NS5206 and NS5217 have low efficacy and only attenuate desensitization partially; (3) NS1493 has low efficacy but nearly completely blocks receptor desensitization. A hydrophobic substituent at the 3-position of the 1,1-dioxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine ring system is important for compound efficacy, with the following ranking: norbornenyl (bicyclo[2.2.1]hept-2-ene) > cyclopentyl > methyl. The replacement of the norbornenyl moiety with a significantly less hydrophobic cyclopentane ring increases the flexibility of the modulator as the cyclopentane ring adopts various conformations at the iGluR2 allosteric binding site. The main structural feature responsible for a nearly complete block of desensitization is the presence of an NH hydrogen bond donor in the 4-position of the 1,1-dioxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine ring system, forming an anchoring hydrogen bond to Ser754. Therefore, the atom at the 4-position of CTZ seems to be a major determinant of receptor desensitization kinetics.