Translation initiation on eukaryotic mRNAs usually occurs by 5'-processive scanning of 40S ribosome subunits from the m7GTP-cap to the initiating AUG. In contrast, picornavirus and some specialized mRNAS initiate translation by internally binding ribosomes. A poorly described third mechanism of initiation, referred to as ribosome shunting or jumping, involves discontinuous scanning by 40S ribosome subunits, in which large segments of the 5' noncoding region are bypassed. Ribosome shunting has only been observed to date on a cauliflower mosaic virus mRNA. In this report we show that the family of adenovirus late mRNAs, which are preferentially translated during infection, use a ribosome jumping mechanism to initiate protein synthesis. Late adenovirus mRNAs contain a common 5'-noncoding region known as the tripartite leader, which confers preferential translation by reducing the requirement for the rate-limiting initiation factor eIF-4F (cap-binding protein complex). Adenovirus inhibits cell protein synthesis largely by inactivating eIF-4F. We show that the tripartite leader directs both 5' linear ribosome scanning and ribosome jumping when eIF-4F is abundant but exclusively uses a ribosome jumping mechanism during late adenovirus infection or heat shock (stress) of mammalian cells, when eIF-4F is altered or inactivated. Shunting is directed by a complex group of secondary structures in the tripartite leader and is facilitated by one or more unidentified viral late gene products. We propose that shunting may represent a widespread mechanism to facilitate selective translation of specialized classes of capped mRNAs, including some stress and developmentally regulated mRNAs, which possess little requirement for eIF-4F but do not initiate by internal ribosome binding.

Baicalin, a flavonoid derived from Scutellaria baicalensis, is the main metabolite of baicalein released following administration in different animal models and human. We previously reported the antiviral activity of baicalein against dengue virus (DENV). Here, we examined the anti-DENV properties of baicalin in vitro, and described the inhibitory potentials of baicalin at different steps of DENV-2 (NGC strain) replication. Our in vitro antiviral experiments showed that baicalin inhibited virus replication at IC50 = 13.5 ± 0.08 μg/ml with SI = 21.5 following virus internalization by Vero cells. Baicalin exhibited virucidal activity against DENV-2 extracellular particles at IC50 = 8.74 ± 0.08 μg/ml and showed anti-adsorption effect with IC50 = 18.07 ± 0.2 μg/ml. Our findings showed that baicalin as the main metabolite of baicalein exerting in vitro anti-DENV activity. Further investigations on baicalein and baicalin to deduce its antiviral therapeutic effects are warranted.

Reverse genetics is a powerful tool to study single-stranded RNA viruses. Despite tremendous efforts having been made to improve the methodology for constructing flavivirus cDNAs, the cause of toxicity of flavivirus cDNAs in bacteria remains unknown. Here we performed mutational analysis studies to identify Escherichia coli promoter (ECP) sequences within nucleotides (nt) 1 to 3000 of the dengue virus type 2 (DENV2) and Japanese encephalitis virus (JEV) genomes. Eight and four active ECPs were demonstrated within nt 1 to 3000 of the DENV2 and JEV genomes, respectively, using fusion constructs containing DENV2 or JEV segments and empty vector reporter gene Renilla luciferase. The Flavivirus genus consists of more than 70 members that are categorized into several antigenic groups (46). Most flaviviruses are transmitted by mosquito or tick vectors and cause serious human and animal diseases (46). They include dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV). DENV and JEV cause some of the most serious arthropod-borne viral illnesses. There are four different serotypes of dengue virus, DENV1, DENV2, DENV3, and DENV4. Dengue cases have been reported in over 100 countries, and an estimated 2.5 billion people live in areas in which dengue is epidemic (26, 27, 49). DENV infection often leads to dengue fever, dengue hemorrhagic fever, and dengue shock syndrome (24,28,48). JEV transmission has been observed in the Southern Hemisphere and has the potential to become a worldwide public health threat. JEV can cause permanent neuropsychiatric sequelae and is sometimes fatal in children (56,60,61).Flaviviruses are enveloped RNA viruses that consist of single-stranded, positive-sense, 10.5-to 11-kb genomic RNA. The genome is associated with multiple copies of capsid proteins that are translated as a single polyprotein. After entering a host cell, the translated polyprotein is then cleaved into three structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by host proteases and a single virus-encoded protease to initiate viral replication (12,18). Introduction of flavivirus genomic RNA into susceptible cell lines can result in the production of infectious virus particles (1). This phenomenon has prompted the study of flavivirus virology via introduction of flavivirus genomic RNA that has been transcribed in vitro from fulllength flavivirus infectious cDNA.Reverse genetics is a powerful method for studying the viral replication of positive-strand RNA viruses (8). Unfortunately, the instability of full-length flavivirus cDNA in Escherichia coli has been a major hurdle in a attempt to construct flavivirus cDNAs (reviewed in references 4, 54, and 63). Several strategies have been developed to avoid or overcome the instability of infectious flavivirus cDNA. The instability of plasmids containing full-length YFV was avoided using an in vitro ligation approach involving two plasmids (5...

Dengue virus (DENV) causes disease globally, with an estimated 25 to 100 million new infections per year. At present, no effective vaccine is available, and treatment is supportive. In this study, we identified BP2109, a potent and selective small-molecule inhibitor of the DENV NS2B/NS3 protease, by a high-throughput screening assay using a recombinant protease complex consisting of the central hydrophilic portion of NS2B and the N terminus of the protease domain. BP2109 inhibited DENV (serotypes 1 to 4), but not Japanese encephalitis virus (JEV), replication and viral RNA synthesis without detectable cytotoxicity. The compound inhibited recombinant DENV-2 NS2B/NS3 protease with a 50% inhibitory concentration (IC 50 ) of 15.43 ؎ 2.12 M and reduced the reporter expression of the DENV-2 replicon with a 50% effective concentration (EC 50 ) of 0.17 ؎ 0.01 M. Sequencing analyses of several individual clones derived from BP2109-resistant DENV-2 RNAs revealed that two amino acid substitutions (R55K and E80K) are found in the region of NS2B, a cofactor of the NS2B/NS3 protease complex. The introduction of R55K and E80K double mutations into the dengue virus NS2B/NS3 protease and a dengue virus replicon construct conferred 10.3-and 73.8-fold resistance to BP2109, respectively. The E80K mutation was further determined to be the key mutation conferring dengue virus replicon resistance (61.3-fold) to BP2109, whereas the R55K mutation alone did not affect resistance to BP2109. Both the R55K and E80K mutations are located in the central hydrophilic portion of the NS2B cofactor, where extensive interactions with the NS3pro domain exist. Thus, our data provide evidence that BP2109 likely inhibits DENV by a novel mechanism.Dengue virus serotypes 1 to 4 (DENV-1 to -4) belongs to the family Flaviviridae, a group of enveloped RNA viruses that includes the genera Hepacivirus, Flavivirus, and Pestivirus. The genus Flavivirus consists of arthropod-borne disease agents, such as the yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and DENV (14). Many members of the genus Flavivirus are important human pathogens and cause significant morbidity and mortality. DENV alone poses a public health threat to an estimated 2.5 billion people living in areas where dengue is epidemic and leads to 50 to 100 million human infections each year (17,18,38). DENV infection often leads to dengue fever, life-threatening dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS) (15,19,37). Approximately 500,000 cases of DHF and DSS have been reported in over 100 countries and have caused approximately 25,000 deaths per year (16). Despite the tremendous efforts invested in anti-DENV research, no clinically approved vaccine or antiviral therapy for humans is available for DENV (25,32,36). Considering the spread of this epidemic and the severity of DENV, the discovery of an effective anti-DENV drug is now an urgent need.DENV is an enveloped RNA virus that consists of a 10.7-kb single-stranded, positive-polarity genomic RN...