Andrew D. Davidson

Dengue Virus Inhibits Alpha Interferon Signaling by Reducing STAT2 Expression

ABSTRACT Alpha/beta interferon (IFN-α/β) is a key mediator of innate antiviral responses but has little effect on the established replication of dengue viruses, which are mosquito-borne flaviviruses of immense global health importance. Understanding how the IFN system is inhibited in dengue virus-infected cells would provide critical insights into disease pathogenesis. In a recent study analyzing the ability of individual dengue virus-encoded proteins to antagonize the IFN response, nonstructural (NS) protein 4B and possibly NS2A and NS4A were identified as candidate IFN antagonists. In monkey cells, NS4B appeared to inhibit both the IFN-α/β and IFN-γ signal transduction pathways, which are distinct but overlapping (J. L. Munoz-Jordan, G. G. Sanchez-Burgos, M. Laurent-Rolle, and A. Garcia-Sastre, Proc. Natl. Acad. Sci. USA 100:14333-14338, 2003). For this study, we examined the effects of dengue virus on the human IFN system, using cell lines that were stably transfected with self-replicating subgenomic dengue virus RNA (replicons) and that expressed all of the dengue virus nonstructural proteins together. We show here that in replicon-containing cells dengue virus RNA replication and the replication of encephalomyocarditis virus, an IFN-sensitive virus, are resistant to the antiviral effects of IFN-α. The presence of dengue virus replicons reduces global IFN-α-stimulated gene expression and specifically inhibits IFN-α but not IFN-γ signal transduction. In cells containing replicons or infected with dengue virus, we found reduced levels of signal transducer and activator of transcription 2 (STAT2), which is a key component of IFN-α but not IFN-γ signaling. Collectively, these data show that dengue virus is capable of subverting the human IFN response by down-regulating STAT2 expression.

Crystal structure of the RNA polymerase domain of the West Nile virus non-structural protein 5

Viruses of the family Flaviviridae are important human and animal pathogens. Among them, the Flaviviruses dengue (DENV) and West Nile (WNV) cause regular outbreaks with fatal outcomes. The RNA-dependent RNA polymerase (RdRp) activity of the non-structural protein 5 (NS5) is a key activity for viral RNA replication. In this study, crystal structures of enzymatically active and inactive WNV RdRp domains were determined at 3.0- and 2.35-Å resolution, respectively. The determined structures were shown to be mostly similar to the RdRps of the Flaviviridae members hepatitis C and bovine viral diarrhea virus, although with unique elements characteristic for the WNV RdRp. Using a reverse genetic system, residues involved in putative interactions between the RNA-cap methyltransferase (MTase) and the RdRp domain of Flavivirus NS5 were identified. This allowed us to propose a model for the structure of the full-length WNV NS5 by in silico docking of the WNV MTase domain (modeled from our previously determined structure of the DENV MTase domain) onto the RdRp domain. The Flavivirus RdRp domain structure determined here should facilitate both the design of anti-Flavivirus drugs and structure-function studies of the Flavivirus replication complex in which the multifunctional NS5 protein plays a central role.

Dengue Virus NS5 Inhibits Interferon-α Signaling by Blocking Signal Transducer and Activator of Transcription 2 Phosphorylation

Type I interferons (interferon [IFN]-alpha/beta) are key mediators of innate antiviral responses. Inhibition of IFN-mediated signal transduction by dengue viruses (DENVs), mosquito-borne flaviviruses of immense global health importance, probably plays a crucial role in determining the outcome of the virus-host interaction. Understanding the molecular basis of IFN antagonism by DENV would therefore provide critical insight into disease pathogenesis and new opportunities for development of antiviral therapies and rationally attenuated vaccines. Here we examine the effects of expression of DENV nonstructural proteins on cellular IFN responses. We show that expression of nonstructural protein 5 (NS5) alone inhibits IFN-alpha, but not IFN-gamma, signaling. Expression of the polymerase domain of NS5 is sufficient to inhibit IFN-alpha signaling. NS5 binds signal transducer and activator of transcription 2 (STAT2) and inhibits its phosphorylation. NS5 alone did not, however, induce degradation of STAT2, which occurs when all nonstructural proteins are expressed together. We conclude that DENV NS5 is a potent and specific type I IFN antagonist.

Mutagenesis of the Dengue Virus Type 2 NS3 Protein within and outside Helicase Motifs: Effects on Enzyme Activity and Virus Replication

ABSTRACT The protein NS3 of Dengue virus type 2 (DEN-2) is the second largest nonstructural protein specified by the virus and is known to possess multiple enzymatic activities, including a serine proteinase located in the N-terminal region and an NTPase-helicase in the remaining 70% of the protein. The latter region has seven conserved helicase motifs found in all members of the family Flaviviridae. DEN-2 NS3 lacking the proteinase region was synthesized as a fusion protein with glutathione S-transferase in Escherichia coli. The effects of 10 mutations on ATPase and RNA helicase activity were examined. Residues at four sites within enzyme motifs I, II, and VI were substituted, and six sites outside motifs were altered by clustered charged-to-alanine mutagenesis. The mutations were also tested for their effects on virus replication by incorporation into genomic-length cDNA. Two mutations, both in motif I (G198A and K199A) abolished both ATPase and helicase activity. Two further mutations, one in motif VI (R457A,R458A) and the other a clustered charged-to-alanine substitution at R376KNGK380, abolished helicase activity only. No virus was detected for any mutation which prevented helicase activity, demonstrating the requirement of this enzyme for virus replication. The remaining six mutations resulted in various levels of enzyme activities, and four permitted virus replication. For the two nonreplicating viruses encoding clustered changes at R184KR186 and D436GEE439, we propose that the substituted residues are surface located and that the viruses are defective through altered interaction of NS3 with other components of the viral replication complex. Two of the replicating viruses displayed a temperature-sensitive phenotype. One contained a clustered mutation at D334EE336 and grew too poorly for further characterization. However, virus with an M283F substitution in motif II was examined in a temperature shift experiment (33 to 37°C) and showed reduced RNA synthesis at the higher temperature.

Identification of a major determinant of mouse neurovirulence of dengue virus type 2 using stably cloned genomic-length cDNA

A genomic-length cDNA clone corresponding to the RNA of dengue virus type 2 (DEN-2) New Guinea C strain (NGC) was constructed in a low copy number vector. The cloned cDNA was stably propagated in Escherichia coil and designated pDVWS501. RNA transcripts produced in vitro from the cDNA using T7 RNA polymerase yielded infectious virus (MON501) upon electroporation into BHK-21 cells. When compared with parental NGC virus, MON501 replicated to similar levels in Aedes albopictus C6/36 cells and showed similar neurovirulence in suckling mice. In contrast, a second genomic-length cDNA clone (pDVWS310) used as an intermediate in the construction of pDVWS501 produced virus (MON310) that replicated well in C6/36 cells but was not neurovirulent in mice. MON310 contained the prM and E genes of the non-neurovirulent PUO-218 strain in an NGC background. There were seven amino acid differences between the prM and E proteins of MON310 and MON501. The differences were generally conservative, with the exception of E residue 126, which was Glu in MON310 and Lys in MON501. To examine the role of this residue in mouse neurovirulence, substitutions of Glu --> Lys and Lys --> Glu were made in MON310 and MON501, respectively. The properties of these mutants clearly demonstrated that Lys at E residue 126 is a major determinant of DEN-2 mouse neurovirulence.

Nuclear Localization of Dengue Virus Nonstructural Protein 5 Through Its Importin α/β–Recognized Nuclear Localization Sequences is Integral to Viral Infection

Dengue virus nonstructural protein 5 (NS5) is a large multifunctional protein with a central role in viral replication. We previously identified two nuclear localization sequences (NLSs) within the central region of dengue virus type‐2 (DENV‐2) NS5 (‘aNLS’ and ‘bNLS’) that are recognized by the importin α/β and importin β1 nuclear transporters, respectively. Here, we demonstrate the importance of the kinetics of NS5 nuclear localization to virus production for the first time and show that the aNLS is responsible. Site‐specific mutations in the bipartite‐type aNLS or bNLS region were introduced into a reporter plasmid encoding green fluorescent protein fused to the N‐terminus of DENV‐2 NS5, as well as into DENV‐2 genomic length complementary DNA. Mutation of basic residues in the highly conserved region of the bNLS did not affect nuclear import of NS5. In contrast, mutations in either basic cluster of the aNLS decreased NS5 nuclear accumulation and reduced virus production, with the greatest reduction observed for mutation of the second cluster (K387K388K389); mutagenesis of both clusters abolished NS5 nuclear import and DENV‐2 virus production completely. The latter appeared to relate to the impaired ability of virus lacking nuclear‐localizing NS5, as compared with wild‐type virus expressing nuclear‐localizing NS5, to reduce interleukin‐8 production as part of the antiviral response. The results overall indicate that NS5 nuclear localization through the aNLS is integral to viral infection, with significant implications for other flaviviruses of medical importance, such as yellow fever and West Nile viruses.

The nsp9 replicase protein of SARS-coronavirus, structure and functional insights.

As part of a high-throughput structural analysis of SARS-coronavirus (SARS-CoV) proteins, we have solved the structure of the non-structural protein 9 (nsp9). This protein, encoded by ORF1a, has no designated function but is most likely involved with viral RNA synthesis. The protein comprises a single β-barrel with a fold previously unseen in single domain proteins. The fold superficially resembles an OB-fold with a C-terminal extension and is related to both of the two subdomains of the SARS-CoV 3C-like protease (which belongs to the serine protease superfamily). nsp9 has, presumably, evolved from a protease. The crystal structure suggests that the protein is dimeric. This is confirmed by analytical ultracentrifugation and dynamic light scattering. We show that nsp9 binds RNA and interacts with nsp8, activities that may be essential for its function(s).

Flexibility between the Protease and Helicase Domains of the Dengue Virus NS3 Protein Conferred by the Linker Region and Its Functional Implications

The dengue virus (DENV) NS3 protein is essential for viral polyprotein processing and RNA replication. It contains an N-terminal serine protease region (residues 1–168) joined to an RNA helicase (residues 180–618) by an 11-amino acid linker (169–179). The structure at 3.15 Å of the soluble NS3 protein from DENV4 covalently attached to 18 residues of the NS2B cofactor region (NS2B18NS3) revealed an elongated molecule with the protease domain abutting subdomains I and II of the helicase (Luo, D., Xu, T., Hunke, C., Grüber, G., Vasudevan, S. G., and Lescar, J. (2008) J. Virol. 82, 173–183). Unexpectedly, using similar crystal growth conditions, we observed an alternative conformation where the protease domain has rotated by ∼161° with respect to the helicase domain. We report this new crystal structure bound to ADP-Mn2+ refined to a resolution of 2.2 Å. The biological significance for interdomain flexibility conferred by the linker region was probed by either inserting a Gly residue between Glu173 and Pro174 or replacing Pro174 with a Gly residue. Both mutations resulted in significantly lower ATPase and helicase activities. We next increased flexibility in the linker by introducing a Pro176 to Gly mutation in a DENV2 replicon system. A 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis were observed. Our results indicate that the linker region has evolved to an optimum length to confer flexibility to the NS3 protein that is required both for polyprotein processing and RNA replication.

Tetracycline-Inducible Packaging Cell Line for Production of Flavivirus Replicon Particles

ABSTRACT We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 × 109 VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 1010 VLPs per 106 transfected cells. Passaging of VLPs on Vero cells or intracerebral injection into 2- to 4-day-old suckling mice illustrated the complete absence of any infectious Kunjin virus. tetKUNCprME cells were also capable of packaging replicon RNA from closely and distantly related flaviviruses, West Nile virus and dengue virus type 2, respectively. The utility of high-titer KUN replicon VLPs was demonstrated by showing increasing CD8+-T-cell responses to encoded foreign protein with increasing doses of KUN VLPs. A single dose of 2.5 × 107 VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 106 splenocytes in an ex vivo gamma interferon enzyme-linked immunospot assay. The packaging cell line thus represents a significant advance in the development of the noncytopathic Kunjin virus replicon-based gene expression system and may be widely applicable to the basic studies of flavivirus RNA packaging and virus assembly as well as to the development of gene expression systems based on replicons from different flaviviruses.

Chapter 2 New Insights into Flavivirus Nonstructural Protein 5

Disease caused by flavivirus infections is an increasing world health problem. Flavivirus nonstructural protein 5 (NS5) possesses enzymatic activities required for capping and synthesis of the viral RNA genome and is essential for virus replication. NS5 is comprised of two domains. The N-terminal domain binds GTP and can perform two biochemically distinct methylation reactions required for RNA cap formation. The C-terminal domain contains RNA-dependent RNA polymerase activity. As such, NS5 is an interesting target against which antiviral drugs could be developed and research toward this goal has accelerated our understanding of NS5 structure and function in recent years. The production and purification of recombinant versions of either the full-length NS5 or the two individual NS5 domains has led to detailed enzymatic studies on NS5 and the determination of structures of the two NS5 domains. In turn, studies using a combination of structural, biochemical, and reverse genetic approaches are revealing how NS5 performs its multifunctional roles in genome replication. Aside from its localization in the membrane-bound replication complex, NS5 can be found free in the cytoplasm and for some flaviviruses in the nucleus of virus-infected cells. NS5 is phosphorylated which may potentially regulate NS5 function and trafficking. Recently, NS5 of a number of flaviviruses has been shown to interact with cellular pathways involved in the host immune response, suggesting that NS5 may play a role in viral pathogenesis. This chapter reviews recent advances in our understanding of the multifunctional roles played by NS5 in the virus lifecycle.