Together with the NS5 polymerase, the NS3 helicase has a pivotal function in flavivirus RNA replication and constitutes an important drug target. We captured the dengue virus NS3 helicase at several stages along the catalytic pathway including bound to single-stranded (ss) RNA, to an ATP analogue, to a transition-state analogue and to ATP hydrolysis products. RNA recognition appears largely sequence independent in a way remarkably similar to eukaryotic DEAD box proteins Vasa and eIF4AIII. On ssRNA binding, the NS3 enzyme switches to a catalyticcompetent state imparted by an inward movement of the P-loop, interdomain closure and a change in the divalent metal coordination shell, providing a structural basis for RNA-stimulated ATP hydrolysis. These structures demonstrate for the first time large quaternary changes in the flaviviridae helicase, identify the catalytic water molecule and point to a b-hairpin that protrudes from subdomain 2, as a critical element for dsRNA unwinding. They also suggest how NS3 could exert an effect as an RNA-anchoring device and thus participate both in flavivirus RNA replication and assembly.

Dengue fever is an important emerging public health concern, with several million viral infections occurring annually, for which no effective therapy currently exists. The NS3 protein from Dengue virus is a multifunctional protein of 69 kDa, endowed with protease, helicase, and nucleoside 5-triphosphatase (NTPase) activities. Thus, NS3 plays an important role in viral replication and represents a very interesting target for the development of specific antiviral inhibitors. We present the structure of an enzymatically active fragment of the Dengue virus NTPase/helicase catalytic domain to 2.4 Å resolution. The structure is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA. Its C-terminal domain adopts a new fold compared to the NS3 helicase of hepatitis C virus, which has interesting implications for the evolution of the Flaviviridae replication complex. A bound sulfate ion reveals residues involved in the metal-dependent NTPase catalytic mechanism. Comparison with the NS3 hepatitis C virus helicase complexed to single-stranded DNA would place the 3 singlestranded tail of a nucleic acid duplex in the tunnel that runs across the basic face of the protein. A possible model for the unwinding mechanism is proposed.

Flaviviruses are a major cause of infectious disease in humans. Dengue virus causes an estimated 50 million cases of febrile illness each year, including an increasing number of cases of hemorrhagic fever. West Nile virus, which recently spread from the Mediterranean basin to the Western Hemisphere, now causes thousands of sporadic cases of encephalitis annually. Despite the existence of licensed vaccines, yellow fever, Japanese encephalitis and tick-borne encephalitis also claim many thousands of victims each year across their vast endemic areas. Antiviral therapy could potentially reduce morbidity and mortality from flavivirus infections, but no effective drugs are currently available. This article introduces a collection of papers in Antiviral Research on molecular targets for flavivirus antiviral drug design and murine models of dengue virus disease that aims to encourage drug development efforts. After reviewing the flavivirus replication cycle, we discuss the envelope glycoprotein, NS3 protease, NS3 helicase, NS5 methyltransferase and NS5 RNA-dependent RNA polymerase as potential drug targets, with special attention being given to the viral protease. The other viral proteins are the subject of individual articles in the journal. Together, these papers highlight current status of drug discovery efforts for flavivirus diseases and suggest promising areas for further research.

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Dengue virus, a member of the family Flaviviridae of positive-strand RNA viruses, has seven non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. Except for enzymic activities contained within NS3 and NS5, the roles of the other proteins in virus replication and pathogenesis are not well defined. In this study, a physical interaction between NS4B and the helicase domain of NS3 was identified by using a yeast two-hybrid assay. This interaction was further confirmed by biochemical pull-down and immunoprecipitation assays, both with purified proteins and with dengue virus-infected cell lysates. NS4B co-localized with NS3 in the perinuclear region of infected human cells. Furthermore, NS4B dissociated NS3 from single-stranded RNA and consequently enhanced the helicase activity of NS3 in an in vitro unwinding assay. These results suggest that NS4B modulates dengue virus replication via its interaction with NS3. INTRODUCTIONDengue fever and its more severe form, dengue haemorrhagic fever, are mosquito-borne viral diseases that are caused by one of the four antigenically distinct serotypes of Dengue virus, DENV-1-DENV-4. Dengue fever affects 50-100 million people in the tropical and subtropical regions annually (Gubler, 1998(Gubler, , 2002. Contemporary demographical and lifestyle trends, such as population explosion and urbanization, have led to the spread of this disease to nonendemic regions. The pathogenesis of dengue fever remains poorly characterized and there are no antivirals or vaccines available to counter this emerging disease.Dengue virus belongs to the family Flaviviridae that consists of enveloped, positive-sense, single-stranded RNA (ssRNA) viruses, such as those that cause yellow fever, Japanese encephalitis, West Nile fever and hepatitis C. Its RNA genome is encapsulated in an icosahedral nucleocapsid (30 nm) that is enveloped in a lipid bilayer (10 nm) (Kuhn et al., 2002) consisting of the membrane and envelope proteins. The 11 kb, capped RNA genome encodes a single polyprotein that is processed co-and post-translationally by host signalases, as well as the virus-encoded serine protease, into the three structural and seven non-structural proteins (NS) in the order C (Core)-prM (pre-Membrane)-E (Envelope)-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 (Chambers & Rice, 1987;Lindenbach & Rice, 2003).The polymerase, helicase and protease enzymic activities encoded by the dengue virus genome ensure virus replication and polyprotein processing. NS3 (618 aa) is a multifunctional protein with protease, helicase, NTPase and 59-terminal RNA triphosphatase activities (Arias et al., 1993;Benarroch et al., 2004;Falgout et al., 1991;Li et al., 1999;Zhang et al., 1992), whilst NS5 (900 aa) has RNAdependent RNA polymerase and methyltransferase activities (Ackermann & Padmanabhan, 2001;Chu & Westaway, 1987;Egloff et al., 2002;Kapoor et al., 1995;Tan et al., 1996). These two proteins form a functional complex that is vital for flavivirus replication (Brooks et al., 2002;Johansson et al., 2001;Yon et al., 2005). The role...

The antiviral activity of UV-4 was previously demonstrated against dengue virus serotype 2 (DENV2) in multiple mouse models. Herein, step-wise minimal effective dose and therapeutic window of efficacy studies of UV-4B (UV-4 hydrochloride salt) were conducted in an antibody-dependent enhancement (ADE) mouse model of severe DENV2 infection in AG129 mice lacking types I and II interferon receptors. Significant survival benefit was demonstrated with 10–20 mg/kg of UV-4B administered thrice daily (TID) for seven days with initiation of treatment up to 48 h after infection. UV-4B also reduced infectious virus production in in vitro antiviral activity assays against all four DENV serotypes, including clinical isolates. A set of purified enzyme, in vitro, and in vivo studies demonstrated that inhibition of endoplasmic reticulum (ER) α-glucosidases and not the glycosphingolipid pathway appears to be responsible for the antiviral activity of UV-4B against DENV. Along with a comprehensive safety package, these and previously published data provided support for an Investigational New Drug (IND) filing and Phases 1 and 2 clinical trials for UV-4B with an indication of acute dengue disease.

High-risk human papillomaviruses (HPVs) are etiologically linked to human cervical and oral cancers. The E6 and E7 oncoproteins encoded by HPV target host cell tumor suppressor proteins. E6 induces proteolysis of p53 through the ubiquitin-proteasome pathway. Recent studies showed that overexpression of E7 caused proteolytic degradation of the tumor suppressor Rb. However, unlike p53, Rb is not regulated by proteolysis in normal cells. In addition, it was unclear whether in its natural context E7 regulates Rb through the ubiquitinproteasome pathway. Therefore, we sought to determine whether Rb is regulated by the ubiquitin-proteasome pathway in HPV-containing tumor cells. We carried out a detailed analysis in Caski cells, that are derived from HPV-containing cervical cancer tissues. Studies with various protease inhibitors revealed that Rb is regulated speci®cally by the ubiquitin-proteasome pathway in HPV-containing cervical tumor cells. Several inhibitors of the 26S proteasome signi®cantly increased the level of Rb in the Caski cells. Rb controls cell growth by forming complexes with the E2F-family transcription factors. Surprisingly, in spite of a signi®cant accumulation of the hypophosphorylated form of Rb, no Rb/E2F complex was detectable in the proteasome inhibitor treated cells. Further analysis revealed that there was an increased accumulation of the E7 oncoprotein. We showed that the proteasome inhibitors simultaneously blocked the proteolysis of E7 and Rb, suggesting that E7 is also regulated by the ubiquitin-dependent proteolysis in cervical cancer cells. Taken together, this study suggests that targeted inhibition of Rb proteolysis will be required for restoring Rb function in HPV-containing cervical cancer cells. Oncogene (2001) 20, 4740 ± 4749.

We performed a mutational analysis of the NS3 helicase of dengue virus to test insights gleaned from its crystal structure and identified four residues in the full-length protein that severely impaired either its RTPase and ATPase (Arg-457-458, Arg-460, Arg-463) or helicase (Ile-365, Arg-376) activity. Alanine substitution of Lys-396, which is located at the surface of domain II, drastically reduced all three enzymatic activities. Our study points to a pocket at the surface of domain II that may be suitable for the design of allosteric inhibitors.The dengue virus NS3 protein contains protease, RNA helicase, 5Ј-nucleoside triphosphatase (NTPase), and RNA 5Ј-triphosphatase (RTPase) activities (1,2,5,15,20,21). NS3 proteins from the four dengue virus serotypes share a minimum of 67% amino acid sequence identity. The enzymatic activities of NS3 proteins from several members of the Flaviviridae have been studied, including hepatitis C virus (HCV) (9), yellow fever virus (YFV) (19), Japanese encephalitis virus (18,19), and West Nile virus (4). The unwinding of duplex RNA structures yielding individual RNA strands is thought to be required for an efficient viral genomic RNA synthesis by the NS5 RNA-dependent RNA polymerase. The essentiality of the helicase activity of NS3 in viral replication has been demonstrated through site-directed mutagenesis (8, 17); hence, it is an attractive target for the design of antiviral compounds. Two three-dimensional (3D) structures of active flavivirus helicase/ NTPase catalytic domains from dengue virus (23) and yellow fever virus (22), respectively, were recently reported. As with the HCV NS3 helicase (12), the structure can be divided into three domains. The seven sequence motifs characteristic of superfamily 2 helicases (7) are present in domains I and II, situated at the N-terminal end of the protein. The NTPase site resides between these two domains. The C-terminal domain III differs most with its HCV counterpart (22, 23) and was suggested to bind NS5 (10). A tunnel that runs across the interface between domain III and the tip of domains I and II was proposed to accommodate a single-stranded nucleic acid tail along which the enzyme could translocate, following interdomain motions triggered by NTP hydrolysis (22,23). Interestingly, recent studies on HCV helicase (14) suggest that the energy derived from nucleic acid binding may be used to unwind several base pairs at the fork essentially without ATP. However, the unidirectional translocation of the enzyme along a long stretch of DNA is derived from ATP hydrolysis.Matusan et al. (17) reported mutations in the conserved motifs of dengue virus NS3 helicase. Here, based on their evolutionary conservation and structural insights (22, 23), we targeted 14 residues within the NS3 helicase (Fig. 1) using site-directed mutagenesis. The mutant proteins were tested for their involvement in the RNA-stimulated NTPase, RTPase, and double-stranded RNA (dsRNA) unwinding activity. While the truncated carboxyl-terminal two-thirds of NS3 used to dete...