Japanese encephalitis virus enters rat neuroblastoma cells via a pH-dependent, dynamin and caveola-mediated endocytosis pathway.

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus and one of the most common agents of viral encephalitis. The infectious entry process of JEV into host cells remains largely unknown. Here, we present a systemic study concerning the cellular entry mechanism of JEV to B104 rat neuroblastoma cells. It was observed that JEV internalization was inhibited by chloroquine and ammonium chloride, both of which can elevate the pH of acidic organelles. However, JEV entry was not affected by chlorpromazine, overexpression of a dominant-negative form of EPS 15 protein, or silencing of the clathrin heavy chain by small interfering RNA (siRNA). These results suggested that JEV entry depended on the acidic intracellular pH but was independent of clathrin. We found that endocytosis of JEV was dependent on membrane cholesterol and was inhibited by inactivation of caveolin-1 with siRNA or dominant-negative mutants. It was also shown, by using the inhibitor dynasore, the K44A mutant, and specific siRNA, that dynamin was required for JEV entry. Phagocytosis or macropinocytosis did not play a role in JEV internalization. In addition, we showed that JEV entry into the neuroblastoma cells is not virus strain specific by assessing the effect of the pharmacological inhibitors on the internalization of JEV belonging to different genotypes. Taken together, our results demonstrate that JEV enters B104 cells through a dynamin-dependent caveola-mediated uptake with a pH-dependent step, which is distinct from the clathrin-mediated endocytosis used by most flaviviruses.

Association of heat-shock protein 70 with lipid rafts is required for Japanese encephalitis virus infection in Huh7 cells.

Japanese encephalitis virus (JEV) is an enveloped flavivirus and the most common agent of viral encephalitis. It enters cells through receptor-mediated endocytosis and low pH-triggered membrane fusion. Although lipid rafts, cholesterol-enriched lipid-ordered membrane domains, have been shown to participate in JEV entry, the mechanisms of the early events of JEV infection, including the cellular receptors of JEV, remain largely unknown. In the current study, it was demonstrated that heat-shock protein 70 (HSP70), rather than other members of the HSP70 family, was required for JEV entry into a human cell line. Cell-surface expression of HSP70 and a direct interaction between JEV envelope (E) protein and HSP70 were observed. Biochemical fractionation showed that HSP70 clearly migrated into the raft fraction after virus infection and co-fractioned with E protein. Depletion of cholesterol shifted the E protein and HSP70 to a non-raft membrane and decreased JEV entry without affecting virus binding to host cells. Notably, recruitment of HSP70 into lipid rafts was required for activation of the phosphoinositide 3-kinase/Akt signalling pathway in the early stage of JEV infection. These results indicate that lipid rafts facilitate JEV entry, possibly by providing a convenient platform to concentrate JEV and its receptors on the host-cell membrane.

RNA interference effectively degrades mRNA and inhibits protein expression of GBV-C E2 gene in Huh7 cells.

The GB virus C/hepatitis G virus (GBV-C/HGV) is a Flaviviridae member that despite its nonpathogenicity, has become of great interest given that it could inhibit the replication of the human immunodeficiency virus (HIV). Therefore, a better knowledge of the viral protein E2 has become our aim. In this study, a GBV-C model cell system (HuhEG) which expressing a fusion protein of the GBV-C E2 protein and enhanced green fluorescent protein (EGFP) stably was established. And the expression of these proteins was silenced effectively by the two E2 gene-specific siRNAs and an EGFP gene-specific siRNA. This inhibition is sequence-specific and extensive (90%). This HuhEG/specific siRNAs system can provide an approach for investigating the association between GBV-C E2 and HIV replication, which may be of potential value in the development of novel prophylactic or therapeutic agents for HIV infection.

Mammalian cell entry protein of Mycobacterium tuberculosis induces the proinflammatory response in RAW 264.7 murine macrophage-like cells.

Mammalian cell entry (Mce1A) protein of Mycobacterium tuberculosis (Mtb) is involved in bacterial entry and survival in macrophages, which has been shown to induce production of tumor necrosis factor alpha (TNF-alpha). It remains unclear whether and how Mce1A functions upon the type I interleukin-1 receptor-associated protein kinase (IRAK-1) and TNF receptor-associated factor 6 (TRAF-6) of important proinflammatory cytokines. In this study, His-tagged Mce1A was expressed and purified. Also, two pieces of small interfering RNA (siRNA) were designed and synthesized by in vitro transcription, which exhibit specific and efficient silencing effect on mce1a expression. Furthermore, RAW 264.7 murine macrophage-like cells were exposed to His-tagged Mce1A or co-transfected with the Mce1A-expressing plasmid and efficient siRNA, and levels of IRAK-1 and TRAF-6 were then determined by Western blot. We show here that Mce1A induces up-regulations of IRAK-1 and TRAF-6 in macrophages in a dose-dependent manner. The level of Caspase-3 closely related with apoptosis was also determined, whereas no changes were observed. These results indicate that Mtb Mce1A protein induces a proinflammatory response in macrophages.

siRNAs targeting terminal sequences of the SARS-associated coronavirus membrane gene inhibit M protein expression through degradation of M mRNA.

SARS-associated coronavirus (SCoV) M protein plays a key role in viral assembly and budding. Recent studies revealed that M protein could interact with N protein in the Golgi complex. In this study, we showed that SCoV M protein co-localized in the Golgi apparatus with a Golgi vector marker. To study M protein function, three candidate small interfering RNAs (siRNAs) corresponding to M gene sequences were designed, transcribed in vitro, and then tested for their ability to silence M protein expression. The plasmid, pEGFP-M, encoding SCoV M protein as a fusion protein with EGFP, was used for silencing and for reporter gene detection in HEK 293T cells transfected with siRNA constructs. The results showed that the mean green fluorescence intensity and M RNA transcripts were significantly reduced, and that the expression of M glycoprotein was strongly inhibited in those cells co-transfected with M-specific siRNAs. These findings demonstrated that the three M-specific siRNAs were able to specifically and effectively inhibit M glycoprotein expression in cultured cells by blocking the accumulation of mRNA, which provides an approach for studies on the functions of M protein and for the development of novel prophylactic or therapeutic agents for SCoV infection.

Up-regulation of ERK and p38 MAPK signaling pathways by hepatitis C virus E2 envelope protein in human T lymphoma cell line.

Hepatitis C virus (HCV) infection correlates with human immune disorders characterized by abnormal activation and proliferation of lymphocytes. Interaction of HCV major envelope protein E2 with susceptible cells occurs at an early stage of the viral infection. HCV tropism for susceptible cells may elicit cellular signaling events implicated in the viral pathogenicity, and E2 protein is known to be responsible for the tropism. We documented previously that HCV E2 protein was capable of activating extracellular signal-regulated kinase (ERK) in human hepatoma Huh-7 cells. Here, ERK and p38 mitogen-activated protein kinase (MAPK) signaling pathways were investigated in human T lymphoma cell line Molt-4 in response to HCV E2 protein. Binding of HCV E2 protein to Molt-4 cells was detectable, and such interaction was a determinant for recognition and delivery of the E2 signal to intracellular pathways. Activation of ERK and p38 MAPK was specifically induced following the HCV E2-cell interaction. CD81 and low-density lipoprotein receptor (LDLR), proposed cellular receptors for HCV, were expressed naturally on Molt-4 cells. CD81 and LDLR were shown to mediate HCV E2-induced activation of ERK and p38 MAPK. In CD81-deficient U937 cells, levels of ERK and p38 MAPK activation and cell proliferation induced by HCV E2 protein were lower than those in Molt-4 cells. Furthermore, cell proliferation and secretion of interferon-gamma and interleukin-10 by Molt-4 cells were promoted by HCV E2 protein. Therefore, ERK and p38 MAPK signaling pathways were up-regulated by HCV E2 protein without synergetic stimulation, which was accompanied by alterations of cell behavior.

Hepatitis G virus genomic RNA is pathogenic to Macaca mulatta.

AIM: To explore the pathogenicity and infectivity of hepatitis G virus (HGV) by observing replication and expression of the virus, as well as the serological and histological changes of Macaca mulatta infected with HGV genomic RNA or HGV RNA-positive serum. METHODS: Full-length HGV cDNA clone (HGVqz) was constructed and proved to be infectious, from which HGV genomic RNA was transcribed in vitro. Macaca mulatta BY1 was intra-hepatically inoculated with HGV genomic RNA, HGV RNA-positive serum from BY1 was intravenously inoculated into Macaca mulatta BM1, and then BB1 was infected with serum from BM1. Serum and liver tissue were taken regularly, and checked with RT-PCR, in situ hybridization and other immunological, serological, histological assays. RESULTS: Serum HGV RNA was detectable in all the 3 Macaca mulattas, serological and histological examinations showed the experimental animals had slightly elevated alanine transaminase (ALT) and developed HGV viremia during the infectious period. The histology, immunohis-tochemistry, and in situ hybridization in liver tissues of the inoculated animals demonstrated a very mild hepatitis with HGV antigen expression in cytoplasm of hepatocytes. RT-PCR and quantitative PCR results showed that HGV could replicate in liver. CONCLUSION: The genomic RNA from full-length HGV cDNA is infectious to the Macaca mulatta and can cause mild hepatitis. HGV RNA-positive serum, from HGV RNA inoculated Macaca mulatta, is infectious to other Macaca mulattas. Macaca mulatta is susceptible to the inoculated HGV, and therefore can be used as an experimental animal model for the studies of HGV infection and pathogenesis.

In vitro molecular evolution of AL NEIBMs improved immunoglobulin (Ig) binding and antibody detection.

AL (SpA A domain-PpL B3 domain), LD5 (PpL B3 domain-SpA D domain-PpL B3 domain-SpA D domain-PpL B3 domain, L-D-L-D-L) and LD3 (PpL B3 domain-SpA D domain-PpL B3 domain, L-D-L) are novel evolved Ig binding molecules (NEIBMs) derived from the in vitro molecular evolution of combinatorial phage libraries displaying randomly rearranged Ig-binding domains of protein A and protein L. These molecules all showed novel Ig-binding properties of double-site binding to the VH3 and Vκ regions of human Ig Fab and high affinity for human IgM, which enhanced IgM detection in the anti-HCV ELISA assay. In this double-site binding, the A domain binds to the VH3 chain with low affinity. Whether the appropriate mutations in the A domain could improve this binding remains unknown. In this study, four combinatorial phage libraries displaying AL mutants with random mutations at different amino acid positions in the A domain were constructed. Seven AL mutant phages with significantly improved Ig binding activity were obtained from the phage library displaying AL mutants randomly mutated at positions 27 and 34 through human IgM-directed in vitro evolution. Two of the seven prokaryotically expressed AL mutants, AL (VV) and AL (KA), exhibited IgM and IgG binding activities equivalent to those of wild-type AL, whereas other mutants showed attenuated binding. However, after labeling with HRP, AL (VV) and AL (KA) showed improved IgM and IgG binding activity, which significantly improved the detection in the anti-HCV assay. Thus, the present study demonstrates that the binding properties of AL were successfully improved through phage-based molecular evolution, which could substantially contribute to the use of AL in antibody detection, and provides an example of successful protein engineering through in vitro molecular evolution.

Significance of palmitoylation of CD81 on its association with tetraspanin-enriched microdomains and mediating hepatitis C virus cell entry.

CD81, a co-receptor for hepatitis C virus (HCV), is a member of the tetraspanin superfamily and is heavily palmitoylated in the juxtamembrane cysteine residues. Palmitoylation plays an important role in protein-protein interactions and association with cholesterol-rich domains of membranes. In this study, Huh7 cells expressing wild-type or palmitoylation-defective CD81 were generated to analyze whether palmitoylation of CD81 is involved in HCV cell entry. Our data showed that de-palmitoylation of CD81 dramatically reduced its association with tetraspanin CD151, but did not influence CD81 partition in detergent-resistant membranes. Moreover, de-palmitoylated CD81 decreased the host cell susceptibility to HCV. Notably, CD151-specific antibodies and siRNA inhibited HCV cell entry, and detachment of CD81 with CD151 decreased the lateral movement of virus particle/CD81 complex to areas of cell-cell contact. These results suggest that palmitoylation of CD81 should facilitate HCV entry, at least in part, by regulating the association of CD81 with tetraspanin-enriched microdomains.

Function of nonstructural 5A protein of genotype 2a in replication and infection of HCV with gene substitution.

AIM: To explore the function of Nonstructural 5A (NS5A) protein of genotype 2a (JFH1) in the replication and infection of hepatitis C virus (HCV). METHODS: Intergenotypic chimera FL-J6JFH/J4NS5A was constructed by inserting NS5A gene from 1b stain HC-J4 by the overlapping polymerase chain reaction (PCR) method and the restriction enzyme reaction. In vitro RNA transcripts of chimera, prototype J6JFH and negative control J6JFH1 (GND) were prepared and transfected into Huh-7.5 cells with liposomes. Immunofluorescence assay (IFA), fluorescence quantitative PCR and infection assay were performed to determine the protein expression and gene replication in Huh-7.5 cells. RESULTS: The HCV RNA levels in FL-J6JFH/J4NS5A chimera RNA transfected cells were significantly lower than the wild type at any indicated time point (2.58 ± 5.97 × 10(6) vs 4.27 ± 1.72 × 10(4), P = 0.032). The maximal level of HCV RNA in chimera was 5.6 ± 1.8 × 10(4) GE/µg RNA at day 34 after transfection, while the wild type reached a peak level at day 13 which was 126 folds higher (70.65 ± 14.11 × 10(5) vs 0.56 ± 0.90 × 10(5), P = 0.028). HCV proteins could also be detected by IFA in chimera-transfected cells with an obviously low level. Infection assay showed that FL-J6JFH/J4NS5A chimera could produce infectious virus particles, ranging from 10 ± 5 ffu/mL to 78.3 ± 23.6 ffu/mL, while that of FL-J6JFH1 ranged from 5.8 ± 1.5 × 10(2) ffu/mL to 2.5 ± 1.4 × 10(4) ffu/mL. CONCLUSION: JFH1 NS5A might play an important role in the robust replication of J6JFH1. The establishment of FL-J6JFH/J4NS5A provided a useful platform for studying the function of other proteins of HCV.

Persistent replication of the GBV-C subgenomic replicons in Huh7 cells.

Studies of GB virus type C (GBV-C) replication in vitro have been limited because of poor growth of GBV-C in cell culture. In order to address the infection of GBV-C, two GBV-C subgenomic replicons (GBCrepEGFP and GBCrepTNF) were developed from a GBV-C full-length genomic cDNA. The viral replication, protein expression and the production of virus-like particles were evaluated in human hepatoma cell line Huh7. The results showed that the established GBCrepEGFP and GBCrepTNF replicons could be replicated autonomously and expressed in cell culture for at least 2 months and 1 month respectively. The replicon RNA could assemble RNA-containing structures in the HuhEH cells expressing GBV-C structural proteins. It suggests that a cell line supporting the replication of GBV-C was established. This replicon system might be used to understand better the biology of GBV-C.

RNA interference effectively inhibits mRNA accumulation and protein expression of hepatitis C virus core and E2 genes in human cells.

RNA interference (RNAi) has been widely used for the analysis of gene function and represents a new promising approach to develop effective antiviral drugs. In this study, several small interfering RNAs (siRNAs) corresponding to two structural genes (core and E2) of hepatitis C virus (HCV) were designed and in vitro transcribed to explore the possibility of silencing these two genes. The plasmids pEGFP-C and pEGFP-E2, which contain the EGFP reporter gene and the core or E2 gene as silencing targets, were co-transfected with siRNAs into HEK 293T cells. At various time points of post-transfection, core and E2 expression levels were detected by fluorescence microscopy, flow cytometry, Western blotting, and real-time quantitative PCR. The results showed that the mean fluorescence intensity, protein expression, and RNA transcripts of siRNAs transfected cells were significantly reduced. This may provide an approach for the development of novel prophylactic or therapeutic agents for HCV infection.

Silencing of SARS-CoV spike gene by small interfering RNA in HEK 293T cells.

Two candidate small interfering RNAs (siRNAs) corresponding to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike gene were designed and in vitro transcribed to explore the possibility of silencing SARS-CoV S gene. The plasmid pEGFP-optS, which contains the codon-optimized SARS-CoV S gene and expresses spike-EGFP fusion protein (S-EGFP) as silencing target and expressing reporter, was transfected with siRNAs into HEK 293T cells. At various time points of posttransfection, the levels of S-EGFP expression and amounts of spike mRNA transcript were detected by fluorescence microscopy, flow cytometry, Western blot, and real-time quantitative PCR, respectively. The results showed that the cells transfected with pEGFP-optS expressed S-EGFP fusion protein at a higher level compared with those transfected with pEGFP-S, which contains wildtype SARS-CoV spike gene sequence. The green fluorescence, mean fluorescence intensity, and SARS-CoV S RNA transcripts were found significantly reduced, and the expression of SARS-CoV S glycoprotein was strongly inhibited in those cells co-transfected with either EGFP- or S-specific siRNAs. Our findings demonstrated that the S-specific siRNAs used in this study were able to specifically and effectively inhibit SARS-CoV S glycoprotein expression in cultured cells through blocking the accumulation of S mRNA, which may provide an approach for studies on the functions of SARS-CoV S gene and development of novel prophylactic or therapeutic agents for SARS-CoV.