A cis-Acting Viral Protein Is Not Required for the Replication of a Coronavirus Defective-Interfering RNA

AbstractMouse hepatitis virus (MHV), a coronavirus, generates defective-interfering (DI) RNAs of different sizes during passages at high multiplicities of infection. All MHV DI RNAs characterized so far contain an open reading frame (ORF) encoding a fused viral protein; in addition, DI RNAs with a long ORF have a competitive advantage over those with a shorter ORF. These findings suggest that DI RNA replication may require an ORF encoding a cis-acting viral protein. In this study, we used a naturally occurring DI RNA and inserted a 12-nucleotide (nt) amber-mutation linker at various positions to truncate the ORF. Most of the mutants replicated as well as the wild-type DI RNA, irrespective of the presence or absence and the length of the ORF in the RNA. Sequence analysis showed that all of the mutants retained the insertional mutations even after two viral passages in tissue culture, establishing that the mutant DI RNAs replicated. We have further introduced two 3-nucleotide substitutions of the first two AUG codons of the ORF, thus completely closing the ORF. This DI RNA replicated as well as the wild-type DL but, after a single passage, the majority of the mutant RNAs was replaced by recombinant RNAs which contain a restored functional ORF. However, an additional insertion of a 12-nt amber-mutation linker downstream of the AUG substitutions prevented recombination, and the DI RNA still replicated. These data indicate that DI RNA replication does not require a DI-specific ORF encoding cis-acting viral proteins and that a 12-nucleotide insertion could prevent or delay the occurrence of RNA recombination, suggesting the importance of direct or indirect RNA alignment in homologous RNA recombination

Highly Sulfated Forms of Heparin Sulfate Are Involved in Japanese Encephalitis Virus Infection

AbstractJapanese encephalitis virus (JEV) infects a broad range of cell types in vitro, though little is known about the initial events of JEV infection. In the present study, we found that highly sulfated glycosaminoglycans (GAGs) are involved in infection of both neurovirulent (RP-9) and attenuated (RP-2ms) JEV strains. Competition experiments using highly sulfated GAGs, heparin and dextran sulfate, demonstrated an inhibition of JEV's attachment and subsequent infection of BHK-21 cells. Treatment of target cells by a potent sulfation inhibitor, sodium chlorate, greatly reduced viral binding ability as well as infection, suggesting a critical role of GAGs' sulfation status on the cellular surface in JEV infection. This phenomenon was confirmed by the manifestation of a distinct binding efficiency of JEV to the wild-type CHO cell line and its mutants with defects in GAG biosynthesis. We also demonstrated the binding of JEV particles and virus envelope glycoprotein to immobilized heparin beads. Furthermore, the addition of heparin suppressed the cytopathic effects induced by JEV infection in cultured cells. Our results establish that the highly sulfated form of GAGs on cell surfaces plays a determining role in the early stage of in vitro JEV infection

Persistence of Japanese Encephalitis Virus Is Associated with Abnormal Expression of the Nonstructural Protein NS1 in Host Cells

AbstractPersistent infection with Japanese encephalitis virus (JEV) was established in murine neuroblastoma N18 cells, and the persistency has been maintained in cell culture for over 6 months. From the persistently infected cells, a clone named C2-2 was selected and expanded to form a stable cell line. The vast majority of C2-2 cells showed viral protein staining by immunofluorescence and continuously produced low levels of virus (103to 104PFU/ml) without marked cytopathic effects or cyclic variations. In addition to the wild-type viral proteins, truncated forms of the viral nonstructural protein 1 (NS1) as well as its derivative NS1′ were produced in C2-2 cells. Both truncated NS1 and NS1′ contain deletions at their N-termini; however, the analyses by RT–PCR and direct sequencing of the viral RNA failed to detect any truncations or mutations within the NS1 region, suggesting that NS1 truncation was a result of a unique posttranslational proteolytic cleavage of NS1 in the persistently infected cells. Similar but not identical truncation of NS1 was also observed in two other persistently infected cell lines established in Vero and DBT (murine astrocytoma) cells. However, viruses released from C2-2 cells did not produce truncated NS1 upon infection of N18 cells, suggesting that NS1 truncations were the result of virus–cell interaction in persistently infected cells. These data indicate a strong association between abnormal NS1 expression and JEV persistency. A probable involvement of dysfunctional NS1 in the establishment and/or maintenance of JEV persistency in tissue culture is discussed

Dengue Virus Targets the Adaptor Protein MITA to Subvert Host Innate Immunity

Dengue is one of the most important arboviral diseases caused by infection of four serotypes of dengue virus (DEN). We found that activation of interferon regulatory factor 3 (IRF3) triggered by viral infection and by foreign DNA and RNA stimulation was blocked by DEN-encoded NS2B3 through a protease-dependent mechanism. The key adaptor protein in type I interferon pathway, human mediator of IRF3 activation (MITA) but not the murine homologue MPYS, was cleaved in cells infected with DEN-1 or DEN-2 and with expression of the enzymatically active protease NS2B3. The cleavage site of MITA was mapped to LRR↓96G and the function of MITA was suppressed by dengue protease. DEN replication was reduced with overexpression of MPYS but not with MITA, while DEN replication was enhanced by MPYS knockdown, indicating an antiviral role of MITA/MPYS against DEN infection. The involvement of MITA in DEN-triggered innate immune response was evidenced by reduction of IRF3 activation and IFN induction in cells with MITA knockdown upon DEN-2 infection. NS2B3 physically interacted with MITA, and the interaction and cleavage of MITA could be further enhanced by poly(dA:dT) stimulation. Thus, we identified MITA as a novel host target of DEN protease and provide the molecular mechanism of how DEN subverts the host innate immunity

Coronavirus Defective-Interfering RNA as an Expression Vector: The Generation of a Pseudorecombinant Mouse Hepatitis Virus Expressing Hemagglutinin-Esterase

AbstractWe have developed an expression vector system using a defective-interfering (DI) RNA of mouse hepatitis virus (MHV), a prototype coronavirus, to deliver and express a foreign gene in MHV-infected cells. This vector contains an MHV intergenic sequence to promote the expression of foreign genes. In this study, we used this vector to introduce a hemagglutininesterase (HE) protein, an optional MHV structural protein, into the MHV-infected cells. The engineered HE protein could be efficiently incorporated into the virion which did not synthesize its own HE protein, thus generating a pseudorecombinant virus that expresses an exogenous HE protein. The engineered HE protein could be made distinguishable from the native protein by attaching an 8-amino-acid peptide tag at the carboxyl-terminus. Both the engineered and native HE proteins from the HE-producing virus train could be incorporated into the virion, thus generating phenotypically mixed virus parficles. We also showed that the HE-expressing DI RNA could be incorporated into viruses, and the engineered HE protein expressed in the infected cells for at least three serial virus passages. Furthermore, we have made two mutants, in which parts of the external domain of the HE protein have been deleted, to study the sequence requirements for the stable expression of HE and its incorporation into MHV virions. Although both of the mutant HE proteins could be expressed in the MHV-infected cells, they failed to be incorporated into virions, suggesting the importance of the extracellular domain of HE protein for its incorporation into virus particles. This vector system enabled the first successful incorporation of a selected coronaviral protein into virions and demonstrates its utility as an expression vector for studying the molecular biology of coronaviruses

Human Kinase/Phosphatase-Wide RNAi Screening Identified Checkpoint Kinase 2 as a Cellular Factor Facilitating Japanese Encephalitis Virus Infection

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes acute encephalitis in humans with high mortality. Not much is known about the interactions between viral and cellular factors that regulate JEV infection. By using a kinase/phosphatase-wide RNAi screening approach, we identified a cell cycle-regulating molecule, checkpoint kinase 2 (CHK2), that plays a role in regulating JEV replication. JEV infection induced G1 arrest and activated CHK2. Inactivation of CHK2 and its upstream ataxia-telangiectasia mutated kinase in JEV-infected cells by using inhibitors reduced virus replication. Likewise, JEV replication was significantly decreased by knockdown of CHK2 expression with shRNA-producing lentiviral transduction. We identified CHK2 as a cellular factor participating in JEV replication, for a new strategy in addressing JEV infection

Cholesterol Effectively Blocks Entry of Flavivirus ▿

Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DEN-2) are enveloped flaviviruses that enter cells through receptor-mediated endocytosis and low pH-triggered membrane fusion and then replicate in intracellular membrane structures. Lipid rafts, cholesterol-enriched lipid-ordered membrane domains, are platforms for a variety of cellular functions. In this study, we found that disruption of lipid raft formation by cholesterol depletion with methyl-β-cyclodextrin or cholesterol chelation with filipin III reduces JEV and DEN-2 infection, mainly at the intracellular replication steps and, to a lesser extent, at viral entry. Using a membrane flotation assay, we found that several flaviviral nonstructural proteins are associated with detergent-resistant membrane structures, indicating that the replication complex of JEV and DEN-2 localizes to the membranes that possess the lipid raft property. Interestingly, we also found that addition of cholesterol readily blocks flaviviral infection, a result that contrasts with previous reports of other viruses, such as Sindbis virus, whose infectivity is enhanced by cholesterol. Cholesterol mainly affected the early step of the flavivirus life cycle, because the presence of cholesterol during viral adsorption greatly blocked JEV and DEN-2 infectivity. Flavirial entry, probably at fusion and RNA uncoating steps, was hindered by cholesterol. Our results thus suggest a stringent requirement for membrane components, especially with respect to the amount of cholesterol, in various steps of the flavivirus life cycle