Proteomics analysis of EV71-infected cells reveals the involvement of host protein NEDD4L in EV71 replication.

Enterovirus 71 (EV71) is a human enterovirus that has seriously affected the Asia-Pacific area for the past two decades. EV71 infection can result in mild hand-foot-and-mouth disease and herpangina and may occasionally lead to severe neurological complications in children. However, the specific biological processes that become altered during EV71 infection remain unclear. To further explore host responses upon EV71 infection, we identified proteins differentially expressed in EV71-infected human glioblastoma SF268 cells using isobaric mass tag (iTRAQ) labeling coupled with multidimensional liquid chromatography-mass spectrometry (LC-MS/MS). Network analysis of proteins altered in cells infected with EV71 revealed that the changed biological processes are related to protein and ion transport, regulation of protein degradation, and homeostatic processes. We confirmed that the levels of NEDD4L and PSMF1 were increased and reduced, respectively, in EV71-infected cells compared to mock-infected control cells. To determine the physiological relevance of our findings, we investigated the consequences of EV71 infection in cells with NEDD4L or PSMF1 depletion. We found that the depletion of NEDD4L significantly reduced the replication of EV71, whereas PSMF1 knockdown enhanced EV71 replication. Collectively, our findings provide the first evidence of proteome-wide dysregulation by EV71 infection and suggest a novel role for the host protein NEDD4L in the replication of this virus.

A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells.

The roles of virus-derived small RNAs (vsRNAs) have been studied in plants and insects. However, the generation and function of small RNAs from cytoplasmic RNA viruses in mammalian cells remain unexplored. This study describes four vsRNAs that were detected in enterovirus 71-infected cells using next-generation sequencing and northern blots. Viral infection produced substantial levels (>10(5) copy numbers per cell) of vsRNA1, one of the four vsRNAs. We also demonstrated that Dicer is involved in vsRNA1 generation in infected cells. vsRNA1 overexpression inhibited viral translation and internal ribosomal entry site (IRES) activity in infected cells. Conversely, blocking vsRNA1 enhanced viral yield and viral protein synthesis. We also present evidence that vsRNA1 targets stem-loop II of the viral 5' untranslated region and inhibits the activity of the IRES through this sequence-specific targeting. Our study demonstrates the ability of a cytoplasmic RNA virus to generate functional vsRNA in mammalian cells. In addition, we also demonstrate a potential novel mechanism for a positive-stranded RNA virus to regulate viral translation: generating a vsRNA that targets the IRES.

Cytoplasmic viral RNA-dependent RNA polymerase disrupts the intracellular splicing machinery by entering the nucleus and interfering with Prp8.

The primary role of cytoplasmic viral RNA-dependent RNA polymerase (RdRp) is viral genome replication in the cellular cytoplasm. However, picornaviral RdRp denoted 3D polymerase (3D(pol)) also enters the host nucleus, where its function remains unclear. In this study, we describe a novel mechanism of viral attack in which 3D(pol) enters the nucleus through the nuclear localization signal (NLS) and targets the pre-mRNA processing factor 8 (Prp8) to block pre-mRNA splicing and mRNA synthesis. The fingers domain of 3D(pol) associates with the C-terminal region of Prp8, which contains the Jab1/MPN domain, and interferes in the second catalytic step, resulting in the accumulation of the lariat form of the splicing intermediate. Endogenous pre-mRNAs trapped by the Prp8-3D(pol) complex in enterovirus-infected cells were identified and classed into groups associated with cell growth, proliferation, and differentiation. Our results suggest that picornaviral RdRp disrupts pre-mRNA splicing processes, that differs from viral protease shutting off cellular transcription and translation which contributes to the pathogenesis of viral infection.

MDA5 plays a crucial role in enterovirus 71 RNA-mediated IRF3 activation.

Induction of type-I interferons (IFNs), IFN-α/ß, is crucial to innate immunity against RNA virus infection. Cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors, including RIG-I and melanoma differentiation-associated gene 5 (MDA5), are critical pathogen sensors for activation of type-I IFN expression in response to RNA virus infection. MDA5 is required for type-I IFN expression in mouse models in response to infection by picornaviruses, such as encephalomyocarditis virus (EMCV) and coxsackievirus B3. Enterovirus 71 (EV71) belongs to picornaviridae and contains positive-stranded RNA genome that is linked with VPg protein at the 5' end. Although a recent study showed that EV71 3C protease could suppress RIG-I-mediated IFN-ß response, the cytoplasmic RIG-I-like receptor that is directly involved in the recognition of EV71 RNA remains unclear. Using EV71-derived RNA as an agonist, we demonstrate that MDA5 is involved in EV71 RNA-mediated IRF3 activation and IFN-ß transcription. Our data also show that overexpression of the MDA5 protein reverses the suppression of IRF3 activation caused by EV71 infection. These results indicate that MDA5 is an important factor for EV71 RNA-activated type-I IFN expression. Furthermore, we also show that EV71 infection enhances MDA5 degradation and that the degradation could be inhibited by a broad spectrum caspase inhibitor.

Proteomic profiling of the 11-dehydrosinulariolide-treated oral carcinoma cells Ca9-22: effects on the cell apoptosis through mitochondrial-related and ER stress pathway.

An oral squamous cell carcinoma Ca9-22 cell line was treated with 11-dehydrosinulariolide, an active compound isolated from the soft coral Sinularia leptoclados, in order to evaluate the effect of this compound on cell growth and protein expression. Cell proliferation was strongly inhibited by 11-dehydrosinulariolide treatment. The 2-DE master maps of control and treated Ca9-22 cells were generated by analysis with the PDQuest software. The comparison between such maps showed up- and down-regulation of 23 proteins, of which 14 were upregulated and 9 were downregulated. The proteomic studies described here have identified some proteins, which are involved in the mitochondrial dysfunction and ER-stress pathway and imply that 11-dehydrosinulariolide induces cell apoptosis through either mitochondrial dysfunction-related or ER stress pathway. Based on this observation, several proteins related to apoptosis pathway were explored for the potential roles involved in this drug-induced cytotoxicity. Furthermore, Salubrinal, an ER stress inhibitor, is able to protect the cell from 11-dehydrosinulariolide-induced apoptosis in a physiological dosage. The significance of these studies illustrates the potential development of anticancer drugs from the natural derivatives of soft coral.

DnaJ homolog Hdj2 facilitates Japanese encephalitis virus replication.

BACKGROUND: Japanese encephalitis virus (JEV) is a member of the mosquito-borne Flaviviridae family of viruses that causes human encephalitis. Upon infection of a new host, replication of viral RNA involves not only the viral RNA-dependent RNA polymerase (RdRp), but also host proteins. Host factors involved in JEV replication are not well characterized. RESULTS: We identified Hdj2, a heat-shock protein 40 (Hsp40)/DnaJ homolog, from a mouse brain cDNA library interacting with JEV nonstructural protein 5 (NS5) encoding viral RdRp using yeast two-hybrid system. Specific interaction of Hdj2 with NS5 was confirmed by coimmunoprecipitation and colocalization in JEV-infected cells. Overexpression of Hdj2 in JEV-infected cells led to an increase of RNA synthesis, and the virus titer was elevated approximately 4.5- to 10-fold. Knocking down of Hdj2 by siRNA reduced the virus production significantly. CONCLUSIONS: We conclude that Hdj2 directly associates with JEV NS5 and facilitates viral replication. This study is the first to demonstrate Hdj2 involved in JEV replication, providing insight into a potential therapeutic target and cell-based vaccine development of JEV infection.

Glyceraldehyde 3-phosphate dehydrogenase negatively regulates the replication of Bamboo mosaic virus and its associated satellite RNA.

The identification of cellular proteins associated with virus replicase complexes is crucial to our understanding of virus-host interactions, influencing the host range, replication, and virulence of viruses. A previous in vitro study has demonstrated that partially purified Bamboo mosaic virus (BaMV) replicase complexes can be employed for the replication of both BaMV genomic and satellite BaMV (satBaMV) RNAs. In this study, we investigated the BaMV and satBaMV 3' untranslated region (UTR) binding proteins associated with these replicase complexes. Two cellular proteins with molecular masses of ∼35 and ∼55 kDa were specifically cross-linked with RNA elements, whereupon the ∼35-kDa protein was identified as the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Gel mobility shift assays confirmed the direct interaction of GAPDH with the 3' UTR sequences, and competition gel shift analysis revealed that GAPDH binds preferentially to the positive-strand BaMV and satBaMV RNAs over the negative-strand RNAs. It was observed that the GAPDH protein binds to the pseudoknot poly(A) tail of BaMV and stem-loop-C poly(A) tail of satBaMV 3' UTR RNAs. It is important to note that knockdown of GAPDH in Nicotiana benthamiana enhances the accumulation of BaMV and satBaMV RNA; conversely, transient overexpression of GAPDH reduces the accumulation of BaMV and satBaMV RNA. The recombinant GAPDH principally inhibits the synthesis of negative-strand RNA in exogenous RdRp assays. These observations support the contention that cytosolic GAPDH participates in the negative regulation of BaMV and satBaMV RNA replication.

Japanese encephalitis virus co-opts the ER-stress response protein GRP78 for viral infectivity.

The serum-free medium from Japanese encephalitis virus (JEV) infected Baby Hamster Kidney-21 (BHK-21) cell cultures was analyzed by liquid chromatography tandem mass spectrometry (LC-MS) to identify host proteins that were secreted upon viral infection. Five proteins were identified, including the molecular chaperones Hsp90, GRP78, and Hsp70. The functional role of GRP78 in the JEV life cycle was then investigated. Co-migration of GRP78 with JEV particles in sucrose density gradients was observed and co-localization of viral E protein with GRP78 was detected by immunofluorescence analysis in vivo. Knockdown of GRP78 expression by siRNA did not effect viral RNA replication, but did impair mature viral production. Mature viruses that do not co-fractionate with GPR78 displayed a significant decrease in viral infectivity. Our results support the hypothesis that JEV co-opts host cell GPR78 for use in viral maturation and in subsequent cellular infections.

A temperature sensitive mutant of heat shock protein 70 reveals an essential role during the early steps of tombusvirus replication.

By co-opting host proteins for their replication, plus-stranded RNA viruses can support robust replication and suppress host anti-viral responses. Tomato bushy stunt virus (TBSV) recruit the cellular heat shock protein 70 (Hsp70), an abundant cytosolic chaperone, into the replicase complex. By taking advantage of yeast model host, we demonstrate that the four-member SSA subfamily of HSP70 genes is essential for TBSV replication. The constitutively expressed SSA1 and SSA2, which are resident proteins in the viral replicase, can be complemented by the heat-inducible SSA3 and/or SSA4 for TBSV replication. Using a yeast strain carrying a temperature sensitive ssa1(ts), but lacking functional SSA2/3/4, we show that inactivation of Ssa1p(ts) led to a defect in membrane localization of the viral replication proteins, resulting in cytosolic distribution of the viral proteins and lack of replicase activity. An in vitro replicase assembly assay with Ssa1p(ts) revealed that functional Ssa1p is required during the replicase assembly process, but not during minus- or plus-strand synthesis. Temperature shift experiments from nonpermissive to permissive in ssa1(ts) yeast revealed that the re-activated Ssa1p(ts) could promote efficient TBSV replication in the absence of other SSA genes. We also demonstrate that the purified recombinant Ssa3p can facilitate the in vitro assembly of the TBSV replicase on yeast membranes, demonstrating that Ssa3p can fully complement the function of Ssa1p. Taken together, the cytosolic SSA subfamily of Hsp70 proteins play essential and multiple roles in TBSV replication.

A key role for heat shock protein 70 in the localization and insertion of tombusvirus replication proteins to intracellular membranes.

Plus-stranded RNA viruses coopt host proteins to promote their robust replication in infected hosts. Tomato bushy stunt tombusvirus (TBSV) is a model virus that can replicate a small replicon RNA in Saccharomyces cerevisiae and in plants. The tombusvirus replicase complex contains heat shock protein 70 (Hsp70), an abundant cytosolic chaperone, which is required for TBSV replication. To dissect the function of Hsp70 in TBSV replication, in this paper we use an Hsp70 mutant (ssa1 ssa2) yeast strain that supports a low level of TBSV replication. Using confocal laser microscopy and cellular fractionation experiments, we find that the localization of the viral replication proteins changes to the cytosol in the mutant cells from the peroxisomal membranes in wild-type cells. An in vitro membrane insertion assay shows that Hsp70 promotes the integration of the viral replication proteins into subcellular membranes. This step seems to be critical for the assembly of the viral replicase complex. Using a gene-silencing approach and quercetin as a chemical inhibitor to downregulate Hsp70 levels, we also confirm the significance of cytosolic Hsp70 in the replication of TBSV and other plant viruses in a plant host. Taken together, our results suggest that cytosolic Hsp70 plays multiple roles in TBSV replication, such as affecting the subcellular localization and membrane insertion of the viral replication proteins as well as the assembly of the viral replicase.

Tomato bushy stunt virus co-opts the RNA-binding function of a host metabolic enzyme for viral genomic RNA synthesis.

Tomato bushy stunt virus (TBSV), a plus-stranded [(+)] RNA plant virus, incorporates the host metabolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) into the viral replicase complex. Here, we show that, during TBSV replication in yeast, the yeast GAPDH Tdh2p moves from the cytosol to the peroxisomal membrane surface, the site of viral RNA synthesis. In yeast cells lacking Tdh2p, decreasing the levels of its functionally redundant homolog Tdh3p inhibited TBSV replication and resulted in equivalent levels of (+) and minus-stranded [(-)] viral RNA, in contrast to the hallmark excess of (+)RNA. Tdh2p specifically bound an AU pentamer sequence in the (-)RNA, suggesting that GAPDH promotes asymmetric RNA synthesis by selectively retaining the (-)RNA template in the replicase complex. Downregulation of GAPDH in a natural plant host decreased TBSV genomic RNA accumulation. Thus, TBSV co-opts the RNA-binding function of a metabolic protein, helping convert the host cell into a viral factory.

Reduced secretion of virions and hepatitis B virus (HBV) surface antigen of a naturally occurring HBV variant correlates with the accumulation of the small S envelope protein in the endoplasmic reticulum and Golgi apparatus.

We identified two novel naturally occurring mutations (W74L and L77R) in the small S envelope protein of hepatitis B virus (HBV). Mutation L77R alone resulted in >10-fold-reduced secretion of virions. In addition, the 2.8-fold reduction of the extracellular HBV surface antigen (HBsAg) of mutant L77R from transfected Huh7 cells appeared to be correlated with a 1.7-fold reduction of intracellular HBsAg, as measured by enzyme-linked immunosorbent assay (ELISA). Surprisingly, opposite to the ELISA results, Western blot analysis revealed a near-10-fold-increased level of the intracellular mutant small S envelope protein. The discrepancy between ELISA and Western blot data was due to significant accumulation of the mutant L77R HBsAg in the intracellular pellet fraction. In contrast to HBsAg, the secretion of HBeAg was normal in L77R-transfected cells. The wild-type HBsAg was usually more diffuse and evenly distributed in the cytoplasm, often outside the perinuclear endoplasmic reticulum (ER) and Golgi apparatus, as observed by immunofluorescence assay. In contrast, the L77R mutant HBsAg tends to be highly restricted within the ER and Golgi, often accumulated in the Golgi compartments distal from the nucleus. The almost exclusive retention in the ER-Golgi of L77R HBsAg was similar to what was observed when the large envelope protein was overexpressed. These multiple aberrant phenotypes of mutant L77R can be corrected by a second naturally occurring S envelope mutation, W74L. Despite the accumulation of L77R HBsAg in ER-Golgi of transfected Huh7 cells, we detected no increase in Grp78 mRNA and proteins, which are common markers for ER stress response.

Hepatocyte-like cells transdifferentiated from a pancreatic origin can support replication of hepatitis B virus.

Recently, a rat pancreatic cell line (AR42J-B13) was shown to transdifferentiate to hepatocyte-like cells upon induction with dexamethasone (Dex). The aim of this study is to determine whether transdifferentiated hepatocytes can indeed function like bona fide liver cells and support replication of hepatotropic hepatitis B virus (HBV). We stably transfected AR42J-B13 cells with HBV DNA and examined the expression of hepatocyte markers and viral activities in control and transdifferentiated cells. A full spectrum of HBV replicative intermediates, including covalently closed circular DNA (cccDNA) and Dane particles, were detected only after induction with Dex and oncostatin M. Strikingly, the small envelope protein and RNA of HBV were increased by 40- to 100-fold upon induction. When HBV RNAs were examined by primer extension analysis, novel core- and precore-specific transcripts were induced by Dex which initiated at nucleotide (nt) 1820 and nt 1789, respectively. Most surprisingly, another species of core-specific RNA, which initiates at nt 1825, is always present at almost equal intensity before and after Dex treatment, a result consistent with Northern blot analysis. The fact that HBV core protein is dramatically produced only after transdifferentiation suggests the possibility of both transcriptional and translational regulation of HBV core antigen in HBV-transfected AR42J-B13 cells. Upon withdrawal of Dex, HBV replication and gene expression decreased rapidly-less than 50% of the cccDNA remained detectable in 1.5 days. Our studies demonstrate that the transdifferentiated AR42J-B13 cells can function like bona fide hepatocytes. This system offers a new opportunity for basic research of virus-host interactions and pancreatic transdifferentiation.