Development and characterization of type I interferon receptor knockout sheep: A model for viral immunology and reproductive signaling.

Type I interferons (IFNs) initiate immune responses to viral infections. Their effects are mediated by the type I IFN receptor, IFNAR, comprised of two subunits: IFNAR1 and IFNAR2. One or both chains of the sheep IFNAR were disrupted in fetal fibroblast lines using CRISPR/Cas9 and 12 lambs were produced by somatic cell nuclear transfer (SCNT). Quantitative reverse transcription-polymerase chain reaction for IFN-stimulated gene expression showed that IFNAR deficient sheep fail to respond to IFN-alpha. Furthermore, fibroblast cells from an IFNAR2 -/- fetus supported significantly higher levels of Zika virus (ZIKV) replication than wild-type fetal fibroblast cells. Although many lambs have died from SCNT related problems or infections, one fertile IFNAR2 -/- ram lived to over 4 years of age, remained healthy, and produced more than 80 offspring. Interestingly, ZIKV infection studies failed to demonstrate a high level of susceptibility. Presumably, these sheep compensated for a lack of type I IFN signaling using the type II, IFN-gamma and type III, IFN-lambda pathways. These sheep constitute a unique model for studying the pathogenesis of viral infection. Historical data supports the concept that ruminants utilize a novel type I IFN, IFN-tau, for pregnancy recognition. Consequently, IFNAR deficient ewes are likely to be infertile, making IFNAR knockout sheep a valuable model for studying pregnancy recognition. A breeding herd of 32 IFNAR2 +/- ewes, which are fertile, has been developed for production of IFNAR2 -/- sheep for both infection and reproduction studies.

Development, Characterization, and Application of Two Reporter-Expressing Recombinant Zika Viruses.

Zika virus (ZIKV), a mosquito-borne transplacentally transmissible flavivirus, is an enveloped virus with an ~10.8 kb plus-strand RNA genome that can cause neurological disease. To facilitate the identification of potential antivirals, we developed two reporter-expressing ZIKVs, each capable of expressing an enhanced green fluorescent protein or an improved luminescent NanoLuc luciferase. First, a full-length functional ZIKV cDNA clone was engineered as a bacterial artificial chromosome, with each reporter gene under the cap-independent translational control of a cardiovirus-derived internal ribosome entry site inserted downstream of the single open reading frame of the viral genome. Two reporter-expressing ZIKVs were then generated by transfection of ZIKV-susceptible BHK-21 cells with infectious RNAs derived by in vitro run-off transcription from the respective cDNAs. As compared to the parental virus, the two reporter-expressing ZIKVs grew to lower titers with slower growth kinetics and formed smaller foci; however, they displayed a genome-wide viral protein expression profile identical to that of the parental virus, except for two previously unrecognized larger forms of the C and NS1 proteins. We then used the NanoLuc-expressing ZIKV to assess the in vitro antiviral activity of three inhibitors (T-705, NITD-008, and ribavirin). Altogether, our reporter-expressing ZIKVs represent an excellent molecular tool for the discovery of novel antivirals.

Early Events in Japanese Encephalitis Virus Infection: Viral Entry.

Japanese encephalitis virus (JEV), a mosquito-borne zoonotic flavivirus, is an enveloped positive-strand RNA virus that can cause a spectrum of clinical manifestations, ranging from mild febrile illness to severe neuroinvasive disease. Today, several killed and live vaccines are available in different parts of the globe for use in humans to prevent JEV-induced diseases, yet no antivirals are available to treat JEV-associated diseases. Despite the progress made in vaccine research and development, JEV is still a major public health problem in southern, eastern, and southeastern Asia, as well as northern Oceania, with the potential to become an emerging global pathogen. In viral replication, the entry of JEV into the cell is the first step in a cascade of complex interactions between the virus and target cells that is required for the initiation, dissemination, and maintenance of infection. Because this step determines cell/tissue tropism and pathogenesis, it is a promising target for antiviral therapy. JEV entry is mediated by the viral glycoprotein E, which binds virions to the cell surface (attachment), delivers them to endosomes (endocytosis), and catalyzes the fusion between the viral and endosomal membranes (membrane fusion), followed by the release of the viral genome into the cytoplasm (uncoating). In this multistep process, a collection of host factors are involved. In this review, we summarize the current knowledge on the viral and cellular components involved in JEV entry into host cells, with an emphasis on the initial virus-host cell interactions on the cell surface.

Functional Genomics and Immunologic Tools: The Impact of Viral and Host Genetic Variations on the Outcome of Zika Virus Infection.

Zika virus (ZIKV) causes no-to-mild symptoms or severe neurological disorders. To investigate the importance of viral and host genetic variations in determining ZIKV infection outcomes, we created three full-length infectious cDNA clones as bacterial artificial chromosomes for each of three spatiotemporally distinct and genetically divergent ZIKVs: MR-766 (Uganda, 1947), P6-740 (Malaysia, 1966), and PRVABC-59 (Puerto Rico, 2015). Using the three molecularly cloned ZIKVs, together with 13 ZIKV region-specific polyclonal antibodies covering nearly the entire viral protein-coding region, we made three conceptual advances: (i) We created a comprehensive genome-wide portrait of ZIKV gene products and their related species, with several previously undescribed gene products identified in the case of all three molecularly cloned ZIKVs. (ii) We found that ZIKV has a broad cell tropism in vitro, being capable of establishing productive infection in 16 of 17 animal cell lines from 12 different species, although its growth kinetics varied depending on both the specific virus strain and host cell line. More importantly, we identified one ZIKV-non-susceptible bovine cell line that has a block in viral entry but fully supports the subsequent post-entry steps. (iii) We showed that in mice, the three molecularly cloned ZIKVs differ in their neuropathogenicity, depending on the particular combination of viral and host genetic backgrounds, as well as in the presence or absence of type I/II interferon signaling. Overall, our findings demonstrate the impact of viral and host genetic variations on the replication kinetics and neuropathogenicity of ZIKV and provide multiple avenues for developing and testing medical countermeasures against ZIKV.

Zika virus: An emerging flavivirus.

Zika virus (ZIKV) is a previously little-known flavivirus closely related to Japanese encephalitis, West Nile, dengue, and yellow fever viruses, all of which are primarily transmitted by blood-sucking mosquitoes. Since its discovery in Uganda in 1947, ZIKV has continued to expand its geographic range, from equatorial Africa and Asia to the Pacific Islands, then further afield to South and Central America and the Caribbean. Currently, ZIKV is actively circulating not only in much of Latin America and its neighbors but also in parts of the Pacific Islands and Southeast Asia. Although ZIKV infection generally causes only mild symptoms in some infected individuals, it is associated with a range of neuroimmunological disorders, including Guillain-Barré syndrome, meningoencephalitis, and myelitis. Recently, maternal ZIKV infection during pregnancy has been linked to neonatal malformations, resulting in various degrees of congenital abnormalities, microcephaly, and even abortion. Despite its emergence as an important public health problem, however, little is known about ZIKV biology, and neither vaccine nor drug is available to control ZIKV infection. This article provides a brief introduction to ZIKV with a major emphasis on its molecular virology, in order to help facilitate the development of diagnostics, therapeutics, and vaccines.

Zika virus: History, epidemiology, transmission, and clinical presentation.

Zika virus (ZIKV), a mosquito-borne positive-stranded RNA virus of the family Flaviviridae (genus Flavivirus), is now causing an unprecedented large-scale outbreak in the Americas. Historically, ZIKV spread eastward from equatorial Africa and Asia to the Pacific Islands during the late 2000s to early 2010s, invaded the Caribbean and Central and South America in 2015, and reached North America in 2016. Although ZIKV infection generally causes no symptoms or only a mild self-limiting illness, it has recently been linked to a rising number of severe neurological diseases, including microcephaly and Guillain-Barré syndrome. Because of the continuous geographic expansion of both the virus and its mosquito vectors, ZIKV poses a serious threat to public health around the globe. However, there are no vaccines or antiviral therapies available against this pathogen. This review summarizes a fast-growing body of literature on the history, epidemiology, transmission, and clinical presentation of ZIKV and highlights the urgent need for the development of efficient control strategies for this emerging pathogen.

Complete Genome Sequences of Three Historically Important, Spatiotemporally Distinct, and Genetically Divergent Strains of Zika Virus: MR-766, P6-740, and PRVABC-59.

Here, we report the 10,807-nucleotide-long consensus RNA genome sequences of three spatiotemporally distinct and genetically divergent Zika virus strains, with the functionality of their genomic sequences substantiated by reverse genetics: MR-766 (African lineage, Uganda, 1947), P6-740 (Asian lineage, Malaysia, 1966), and PRVABC-59 (Asian lineage-derived American strain, Puerto Rico, 2015).

Comparison of the live-attenuated Japanese encephalitis vaccine SA14-14-2 strain with its pre-attenuated virulent parent SA14 strain: similarities and differences in vitro and in vivo.

Japanese encephalitis virus (JEV) is the main cause of acute viral encephalitis, primarily affecting children and young adults in the Asia-Pacific region. JEV is a vaccine-preventable pathogen, with four types of JE vaccine licensed in different regions of the world. To date, the most common JEV strain used in vaccine development and production is SA14-14-2, an attenuated strain derived from its wild-type parental strain SA14. In this study, we directly compared the phenotypic and genotypic characteristics of SA14 and SA14-14-2 to determine the biological and genetic properties associated with their differential virulence. In susceptible BHK-21 cells, SA14-14-2 grew slightly more slowly and formed smaller plaques than SA14, but unlike SA14, it showed almost no expression of the viral protein NS1', the product of a conserved predicted RNA pseudoknot-mediated ribosomal frameshift. In weanling ICR mice, SA14-14-2 was highly attenuated in terms of both neuroinvasiveness and neurovirulence, with its median lethal doses invariably over five logs higher than those of SA14 when inoculated intramuscularly and intracerebrally. Interestingly, the neurovirulence of SA14-14-2 was dependent on mouse age, with the 1- to 7-day-old mice being highly susceptible and the 14- to 21-day-old mice becoming resistant to intracerebral inoculation. At the genome level, SA14-14-2 differed from SA14 by 57 nucleotides, including one silent G-to-A substitution at position 3599 within the predicted RNA pseudoknot for NS1' synthesis; of the 57 differences, 25 resulted in amino acid substitutions. Our data pave the way for the development of new genetically modified JE vaccines.

Profiling of viral proteins expressed from the genomic RNA of Japanese encephalitis virus using a panel of 15 region-specific polyclonal rabbit antisera: implications for viral gene expression.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is closely related to West Nile (WN), yellow fever (YF), and dengue (DEN) viruses. Its plus-strand genomic RNA carries a single open reading frame encoding a polyprotein that is cleaved into three structural (C, prM/M, and E) and at least seven nonstructural (NS1/NS1', NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins, based on previous work with WNV, YFV, and DENV. Here, we aimed to profile experimentally all the viral proteins found in JEV-infected cells. We generated a collection of 15 JEV-specific polyclonal antisera covering all parts of the viral protein-coding regions, by immunizing rabbits with 14 bacterially expressed glutathione-S-transferase fusion proteins (for all nine viral proteins except NS2B) or with a chemically synthesized oligopeptide (for NS2B). In total lysates of JEV-infected BHK-21 cells, immunoblotting with these antisera revealed: (i) three mature structural proteins (~12-kDa C, ~8-kDa M, and ~53-kDa E), a precursor of M (~24-kDa prM) and three other M-related proteins (~10-14 kDa); (ii) the predicted ~45-kDa NS1 and its frameshift product, ~58-kDa NS1', with no evidence of the predicted ~25-kDa NS2A; (iii) the predicted but hardly detectable ~14-kDa NS2B and an unexpected but predominant ~12-kDa NS2B-related protein; (iv) the predicted ~69-kDa NS3 plus two major cleavage products (~34-kDa NS3N-term and ~35-kDa NS3C-term), together with at least nine minor proteins of ~16-52 kDa; (v) the predicted ~14-kDa NS4A; (vi) two NS4B-related proteins (~27-kDa NS4B and ~25-kDa NS4B'); and (vii) the predicted ~103-kDa NS5 plus at least three other NS5-related proteins (~15 kDa, ~27 kDa, and ~90 kDa). Combining these data with confocal microscopic imaging of the proteins' intracellular localization, our study is the first to provide a solid foundation for the study of JEV gene expression, which is crucial for elucidating the regulatory mechanisms of JEV genome replication and pathobiology.

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses.

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

A molecularly cloned, live-attenuated japanese encephalitis vaccine SA14-14-2 virus: a conserved single amino acid in the ij Hairpin of the Viral E glycoprotein determines neurovirulence in mice.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV represents the JE serogroup, which also includes West Nile, Murray Valley encephalitis, and St. Louis encephalitis viruses. Within this serogroup, JEV is a vaccine-preventable pathogen, but the molecular basis of its neurovirulence remains unknown. Here, we constructed an infectious cDNA of the most widely used live-attenuated JE vaccine, SA14-14-2, and rescued from the cDNA a molecularly cloned virus, SA14-14-2MCV, which displayed in vitro growth properties and in vivo attenuation phenotypes identical to those of its parent, SA14-14-2. To elucidate the molecular mechanism of neurovirulence, we selected three independent, highly neurovirulent variants (LD50, <1.5 PFU) from SA14-14-2MCV (LD50, >1.5×105 PFU) by serial intracerebral passage in mice. Complete genome sequence comparison revealed a total of eight point mutations, with a common single G1708→A substitution replacing a Gly with Glu at position 244 of the viral E glycoprotein. Using our infectious SA14-14-2 cDNA technology, we showed that this single Gly-to-Glu change at E-244 is sufficient to confer lethal neurovirulence in mice, including rapid development of viral spread and tissue inflammation in the central nervous system. Comprehensive site-directed mutagenesis of E-244, coupled with homology-based structure modeling, demonstrated a novel essential regulatory role in JEV neurovirulence for E-244, within the ij hairpin of the E dimerization domain. In both mouse and human neuronal cells, we further showed that the E-244 mutation altered JEV infectivity in vitro, in direct correlation with the level of neurovirulence in vivo, but had no significant impact on viral RNA replication. Our results provide a crucial step toward developing novel therapeutic and preventive strategies against JEV and possibly other encephalitic flaviviruses.

Japanese encephalitis: the virus and vaccines.

Japanese encephalitis (JE) is an infectious disease of the central nervous system caused by Japanese encephalitis virus (JEV), a zoonotic mosquito-borne flavivirus. JEV is prevalent in much of Asia and the Western Pacific, with over 4 billion people living at risk of infection. In the absence of antiviral intervention, vaccination is the only strategy to develop long-term sustainable protection against JEV infection. Over the past half-century, a mouse brain-derived inactivated vaccine has been used internationally for active immunization. To date, however, JEV is still a clinically important, emerging, and re-emerging human pathogen of global significance. In recent years, production of the mouse brain-derived vaccine has been discontinued, but 3 new cell culture-derived vaccines are available in various parts of the world. Here we review current aspects of JEV biology, summarize the 4 types of JEV vaccine, and discuss the potential of an infectious JEV cDNA technology for future vaccine development.

Overview: Replication of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus that causes significant losses in the pig industry, is one of the most important animal pathogens of global significance. Since the discovery of the virus, significant progress has been made in understanding its epidemiology and transmission, but no adequate control measures are yet available to eliminate infection with this pathogen. The genome replication of PRRSV is required to reproduce, within a few hours of infection, the millions of progeny virions that establish, disseminate, and maintain infection. Replication of the viral RNA genome is a multistep process involving a replication complex that is formed not only from components of viral and cellular origin but also from the viral genomic RNA template; this replication complex is embedded within particular virus-induced membrane vesicles. PRRSV RNA replication is directed by at least 14 replicase proteins that have both common enzymatic activities, including viral RNA polymerase, and also unusual and poorly understood RNA-processing functions. In this review, we summarize our current understanding of PRRSV replication, which is important for developing a successful strategy for the prevention and control of this pathogen.

Biological and genetic properties of SA14-14-2, a live-attenuated Japanese encephalitis vaccine that is currently available for humans.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is a major cause of acute encephalitis, a disease of significance for global public health. In the absence of antiviral therapy to treat JEV infection, vaccination is the most effective method of preventing the disease. In JE-endemic areas, the most widely used vaccine to date is SA(14)-14-2, a live-attenuated virus derived from its virulent parent SA(14). In this study, we describe the biological properties of SA(14)-14-2, both in vitro and in vivo, and report the genetic characteristics of its genomic RNA. In BHK-21 (hamster kidney) cells, SA(14)-14-2 displayed a slight delay in plaque formation and growth kinetics when compared to a virulent JEV strain, CNU/LP2, with no decrease in maximum virus production. The delay in viral growth was also observed in two other cell lines, SH-SY5Y (human neuroblastoma) and C6/36 (mosquito larva), which are potentially relevant to JEV pathogenesis and transmission. In 3-week-old ICR mice, SA(14)-14-2 did not cause any symptoms or death after either intracerebral or peripheral inoculation with a maximum dose of up to 1.5×10(3) plaque-forming units (PFU) per mouse. The SA(14)-14-2 genome consisted of 10977 nucleotides, one nucleotide longer than all the previously reported genomes of SA(14)-14-2, SA(14) and two other SA(14)-derived attenuated viruses. This difference was due to an insertion of one G nucleotide at position 10701 in the 3 noncoding region. Also, we noted a significant number of nucleotide and/or amino acid substitutions throughout the genome of SA(14)-14-2, except for the prM protein-coding region, that differed from SA(14) and/or the other two attenuated viruses. Our results, together with others', provide a foundation not only for the study of JEV virulence but also for the development of new and improved vaccines for JEV.

A cross-protective mAb recognizes a novel epitope within the flavivirus NS1 protein.

Despite a resurgence of flavivirus infections worldwide, no approved therapeutic agent exists for any member of the genus. While cross-reactive antibodies with therapeutic potential against flaviviruses have been generated, the majority of them are anti-E antibodies with the potential to cause antibody-dependent enhancement of flavivirus infection and disease. We described previously mAbs against the non-structural NS1 protein of the West Nile virus (WNV) that were protective in mice when administered pre- or post-infection of WNV. Here, we demonstrate that one of these mAbs (16NS1) cross-reacted with Japanese encephalitis virus (JEV) and exhibited protective activity against a lethal JEV infection. Overlapping peptide mapping analysis combined with site-specific mutations identified a novel epitope ¹¹6KAWGKSILFA¹²5 and critical amino acid residues (¹¹8W and ¹²²I) for 16NS1 mAb binding. These results may facilitate the development of a broadly therapeutic mAb that lacks enhancing potential and/or subunit-based vaccine against flaviviruses that target the NS1 protein.

Packaging of porcine reproductive and respiratory syndrome virus replicon RNA by a stable cell line expressing its nucleocapsid protein.

Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the Arteriviridae family, is one of the most common and economically important swine pathogens. Although both live-attenuated and killed-inactivated vaccines against the virus have been available for a decade, PRRSV is still a major problem in the swine industry worldwide. To explore the possibility of producing single-round infectious PRRSV replicon particles as a potential vaccine strategy, we have now generated two necessary components: 1) a stable cell line (BHK/Sinrepl9/PRRSV-N) that constitutively expresses the viral nucleocapsid (N) protein localized to the cytoplasm and the nucleolus and 2) a PRRSV replicon vector (pBAC/PRRSV/Replicon-AN) with a 177-nucleotide deletion, removing the 3'-half portion of ORF7 in the viral genome, from which the self-replicating propagation-defective replicon RNAs were synthesized in vitro by SP6 polymerase run-off transcription. Transfection of this replicon RNA into N protein-expressing BHK-21 cells led to the secretion of infectious particles that packaged the replicon RNA, albeit with a low production efficiency of 0.4 × 10(2) to 1.1 × 10(2) infectious units/ml; the produced particles had only single-round infectivity with no cell-to-cell spread. This trans-complementation system for PRRSV provides a useful platform for studies to define the packaging signals and motifs present within the viral genome and N protein, respectively, and to develop viral replicon-based antiviral vaccines that will stop the infection and spread of this pathogen.

Complete genome sequence and phylogenetic analysis of a recombinant Korean norovirus, CBNU1, recovered from a 2006 outbreak.

We have determined the complete nucleotide and deduced amino acid sequences of the RNA genome of CBNU1, a human norovirus (NoV) recovered from a 2006 outbreak in South Korea. The genome of 7547 nucleotides, excluding a 3'-poly(A) tail of 11-105 nucleotides, encodes three overlapping open reading frames (ORFs): ORF1 (nucleotides 5-5104), ORF2 (nucleotides 5085-6731), and ORF3 (nucleotides 6731-7495). In a comparison to 108 other currently available completely sequenced NoVs representing all five genogroups (GI-GV) except GIV, the CBNU1 strain was highly similar to GII.3 NoVs. Multiple sequence alignments of the completely sequenced NoV genomes revealed five hypervariable regions throughout their genomes: two in ORF1, one in ORF2, and two in ORF3. At both the nucleotide and amino acid levels, genome-based phylogenetic analyses invariably showed that the CBNU1 strain was most closely related to three GII.3 NoVs: the American Texas/TCH04-577 and the two Japanese Saitama U18 and Saitama U201 strains; furthermore, these genome-based phylogenetic topologies corresponded most closely to those based on the ORF2 genes, as compared to those based on the ORF1 and ORF3 genes. Subsequent ORF2-based phylogenetic analyses of a selection of 126 other NoVs representing all 19 GII genotypes, in combination with genome-based Simplot analyses, showed that the CBNU1 strain was a recombinant GII.3 NoV with a breakpoint at the ORF1/ORF2 junction between two putative parent-like strains, Guangzhou/NVgz01 and Texas/TCH04-577. Overall, the CBNU1 strain represents the first Korean human NoV whose genome has been completely sequenced and for which its relationship with a large panel of genetically diverse NoVs has been extensively characterized.

3' cis-acting elements that contribute to the competence and efficiency of Japanese encephalitis virus genome replication: functional importance of sequence duplications, deletions, and substitutions.

The positive-strand RNA genome of Japanese encephalitis virus (JEV) terminates in a highly conserved 3'-noncoding region (3'NCR) of six domains (V, X, I, II-1, II-2, and III in the 5'-to-3' direction). By manipulating the JEV genomic RNA, we have identified important roles for RNA elements present within the 574-nucleotide 3'NCR in viral replication. The two 3'-proximal domains (II-2 and III) were sufficient for RNA replication and virus production, whereas the remaining four (V, X, I, and II-1) were dispensable for RNA replication competence but required for maximal replication efficiency. Surprisingly, a lethal mutant lacking all of the 3'NCR except domain III regained viability through pseudoreversion by duplicating an 83-nucleotide sequence from the 3'-terminal region of the viral open reading frame. Also, two viable mutants displayed severe genetic instability; these two mutants rapidly developed 12 point mutations in domain II-2 in the mutant lacking domains V, X, I, and II-1 and showed the duplication of seven upstream sequences of various sizes at the junction between domains II-1 and II-2 in the mutant lacking domains V, X, and I. In all cases, the introduction of these spontaneous mutations led to an increase in RNA production that paralleled the level of protein accumulation and virus yield. Interestingly, the mutant lacking domains V, X, I, and II-1 was able to replicate in hamster BHK-21 and human neuroblastoma SH-SY5Y cells but not in mosquito C6/36 cells, indicating a cell type-specific restriction of its viral replication. Thus, our findings provide the basis for a detailed map of the 3' cis-acting elements in JEV genomic RNA, which play an essential role in viral replication. They also provide experimental evidence for the function of 3' direct repeat sequences and suggest possible mechanisms for the emergence of these sequences in the 3'NCR of JEV and perhaps in other flaviviruses.

Japanese encephalitis virus-based replicon RNAs/particles as an expression system for HIV-1 Pr55 Gag that is capable of producing virus-like particles.

Ectopic expression of the structural protein Pr55(Gag) of HIV-1 has been limited by the presence of inhibitory sequences in the gag coding region that must normally be counteracted by HIV-1 Rev and RRE. Here, we describe a cytoplasmic RNA replicon based on the RNA genome of Japanese encephalitis virus (JEV) that is capable of expressing HIV-1 gag without requiring Rev/RRE. This replicon system was constructed by deleting all three JEV structural protein-coding regions (C, prM, and E) from the 5'-proximal region of the genome and simultaneously inserting an HIV-1 gag expression cassette driven by the internal ribosome entry site of encephalomyocarditis virus into the 3'-proximal noncoding region of the genome. Transfection of this JEV replicon RNA led to expression of Pr55(Gag) in the absence of Rev/RRE in the cytoplasm of hamster BHK-21, human HeLa, and mouse NIH/3T3 cells. Production of the Pr55(Gag) derived from this JEV replicon RNA appeared to be increased by approximately 3-fold when compared to that based on an alphavirus replicon RNA. Biochemical and morphological analyses demonstrated that the Pr55(Gag) proteins were released into the culture medium in the form of virus-like particles. We also observed that the JEV replicon RNAs expressing the Pr55(Gag) could be encapsidated into single-round infectious JEV replicon particles when transfected into a stable packaging cell line that provided the three JEV structural proteins in trans. This ectopic expression of the HIV-1 Pr55(Gag) by JEV-based replicon RNAs/particles in diverse cell types may represent a useful molecular platform for various biological applications in medicine and industry.

A complex RNA motif defined by three discontinuous 5-nucleotide-long strands is essential for Flavivirus RNA replication.

Tertiary or higher-order RNA motifs that regulate replication of positive-strand RNA viruses are as yet poorly understood. Using Japanese encephalitis virus (JEV), we now show that a key element in JEV RNA replication is a complex RNA motif that includes a string of three discontinuous complementary sequences (TDCS). The TDCS consists of three 5-nt-long strands, the left (L) strand upstream of the translation initiator AUG adjacent to the 5'-end of the genome, and the middle (M) and right (R) strands corresponding to the base of the Flavivirus-conserved 3' stem-loop structure near the 3'-end of the RNA. The three strands are arranged in an antiparallel configuration, with two sets of base-pairing interactions creating L-M and M-R duplexes. Disrupting either or both of these duplex regions of TDCS completely abolished RNA replication, whereas reconstructing both duplex regions, albeit with mutated sequences, fully restored RNA replication. Modeling of replication-competent genomes recovered from a large pool of pseudorevertants originating from six replication-incompetent TDCS mutants suggests that both duplex base-pairing potentials of TDCS are required for RNA replication. In all cases, acquisition of novel sequences within the 3'M-R duplex facilitated a long-range RNA-RNA interaction of its 3'M strand with either the authentic 5'L strand or its alternative (invariably located upstream of the 5' initiator), thereby restoring replicability. We also found that a TDCS homolog is conserved in other flaviviruses. These data suggest that two duplex base-pairings defined by the TDCS play an essential regulatory role in a key step(s) of Flavivirus RNA replication.