Hepatitis C virus down-regulates SERPINE1/PAI-1 expression to facilitate its replication.

Identification of host factors involved in viral replication is critical for understanding the molecular mechanism of viral replication and pathogenesis. Genes differentially expressed in HuH-7 cells with or without a hepatitis C virus (HCV) sub-genomic replicon were screened by microarray analysis. SERPINE1/PAI-1 was found to be down-regulated after HCV infection in this analysis. Down-regulation of SERPINE1/PAI-1 expression at the transcriptional level was verified by the real-time reverse transcriptase (RT)-PCR assay. Reduced SERPINE1/PAI-1 protein secretion was detected in the supernatant of HCV replicon cells and in sera from HCV-infected patients. SERPINE1 gene expression was down-regulated by HCV NS3/4A and NS5A proteins through the transforming growth factor-ß (TGF-ß) signalling pathway at the transcriptional level. Down-regulated genes in HCV replicon cells could be the factors supressing HCV replication. Indeed, over-expressed PAI-1 inhibited HCV replication but the mechanism is unknown. It has been demonstrated that HCV induces the expression of TGF-ß, and TGF-ß enhances HCV replication by a not-yet-defined mechanism. SERPINE1/PAI-1 is also known to be potently induced by TGF-ß at the transcriptional level through both Smad-dependent and Smad-independent pathways. The exogenously expressed SERPINE1/PAI-1 suppressed the expression of the endogenous SERPINE1 gene at the transcriptional level through the TGF-ß signalling but not the Smad pathway. Thus, SERPINE1/PAI-1 could suppress HCV replication possibly by negatively regulating TGF-ß signalling. A model is proposed for the interplay betweenthe TGF-ß signalling pathway, HCV and SERPINE1/PAI-1 to keep the homeostasis of the cells.

Influenza A virus upregulates PRPF8 gene expression to increase virus production.

A ddRT-PCR analysis was performed to detect cellular genes that are differentially expressed after influenza A virus (H1N1) infection of A549 cells. After ddRT-PCR, eight DNA fragments were identified. PRPF8, one of the cellular genes that were upregulated after virus infection, was further analyzed since it has previously been identified as a cellular factor required for influenza virus replication. The upregulation of PRPF8 gene expression after viral infection was confirmed using real-time RT-PCR for mRNA detection and Western blot analysis for protein detection. Influenza A virus also upregulated the PRPF8 promoter in a reporter assay. In addition to H1N1, influenza A virus H3N2 and influenza B virus could also activate PRPF8 expression. Therefore, upregulation of PRPF8 expression might be important for the replication of different influenza viruses. Indeed, overexpression of PRPF8 gene enhanced virus production, while knockdown of expression of this gene reduced viral production significantly. To determine which viral protein could enhance PRPF8 gene expression, individual viral genes were cloned and expressed. Among the different viral proteins, expression of either the viral NS1 or PB1 gene could upregulate the PRPF8 expression. Our results from this study indicate that influenza A virus upregulates cellular PRPF8 gene expression through viral NS1 and PB1 proteins to increase virus production.

Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice.

The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made it more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that ß-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through ß-catenin signaling pathway.

Hepatitis C Virus Down-Regulates the Expression of Ribonucleotide Reductases to Promote Its Replication.

Ribonucleotide reductases (RRs or RNRs) catalyze the reduction of the OH group on the 2nd carbon of ribose, reducing four ribonucleotides (NTPs) to the corresponding deoxyribonucleotides (dNTPs) to promote DNA synthesis. Large DNA viruses, such as herpesviruses and poxviruses, could benefit their replication through increasing dNTPs via expression of viral RRs. Little is known regarding the relationship between cellular RRs and RNA viruses. Mammalian RRs contain two subunits of ribonucleotide reductase M1 polypeptide (RRM1) and two subunits of ribonucleotide reductase M2 polypeptide (RRM2). In this study, expression of cellular RRMs, including RRM1 and RRM2, is found to be down-regulated in hepatitis C virus (HCV)-infected Huh7.5 cells and Huh7 cells with HCV subgenomic RNAs (HCVr). As expected, the NTP/dNTP ratio is elevated in HCVr cells. Compared with that of the control Huh7 cells with sh-scramble, the NTP/dNTP ratio of the RRM-knockdown cells is elevated. Knockdown of RRM1 or RRM2 increases HCV replication in HCV replicon cells. Moreover, inhibitors to RRMs, including Didox, Trimidox and hydroxyurea, enhance HCV replication. Among various HCV viral proteins, the NS5A and/or NS3/4A proteins suppress the expression of RRMs. When these are taken together, the results suggest that HCV down-regulates the expression of RRMs in cultured cells to promote its replication.

SARS-CoV nucleocapsid protein interacts with cellular pyruvate kinase protein and inhibits its activity.

The pathogenesis of SARS-CoV remains largely unknown. To study the function of the SARS-CoV nucleocapsid protein, we have conducted a yeast two-hybrid screening experiment to identify cellular proteins that may interact with the SARS-CoV nucleocapsid protein. Pyruvate kinase (liver) was found to interact with SARS-CoV nucleocapsid protein in this experiment. The binding domains of these two proteins were also determined using the yeast two-hybrid system. The physical interaction between the SARS-CoV nucleocapsid and cellular pyruvate kinase (liver) proteins was further confirmed by GST pull-down assay, co-immunoprecipitation assay and confocal microscopy. Cellular pyruvate kinase activity in hepatoma cells was repressed by SARS-CoV nucleocapsid protein in either transiently transfected or stably transfected cells. PK deficiency in red blood cells is known to result in human hereditary non-spherocytic hemolytic anemia. It is reasonable to assume that an inhibition of PKL activity due to interaction with SARS-CoV N protein is likely to cause the death of the hepatocytes, which results in the elevation of serum alanine aminotransferase and liver dysfunction noted in most SARS patients. Thus, our results suggest that SARS-CoV could reduce pyruvate kinase activity via its nucleocapsid protein, and this may in turn cause disease.

Long noncoding RNAs in hepatitis B virus replication and oncogenesis.

Several diverse long noncoding RNAs (lncRNAs) have been identified to be involved in hepatitis B virus (HBV) replication and oncogenesis, especially those dysregulated in HBV-related hepatocellular carcinoma (HCC). Most of these dysregulated lncRNAs are modulated by the HBV X protein. The regulatory mechanisms of some lncRNAs in HBV replication and oncogenesis have been characterized. Genetic polymorphisms of several lncRNAs affecting HBV replication or oncogenesis have also been studied. The prognosis of HCC remains poor. It is important to identify novel tumor markers for early diagnosis and find more therapeutic targets for effective treatments of HCC. Some dysregulated lncRNAs in HBV-related HCC may become biomarkers for early diagnosis and/or the therapeutic targets of HCC. This mini-review summarizes these findings briefly, focusing on recent developments.

Roles of microRNAs in Hepatitis C Virus Replication and Pathogenesis.

Hepatitis C virus (HCV) infection is associated with the development of chronic liver diseases, e.g., fibrosis, cirrhosis, even hepatocellular carcinoma, and/or extra-hepatic diseases such as diabetes. As an obligatory intracellular pathogen, HCV absolutely relies on host cells to propagate and is able to modulate host cellular factors in favor of its replication. Indeed, lots of cellular factors, including microRNAs (miRNAs), have been identified to be dysregulated during HCV infection. MiRNAs are small noncoding RNAs that regulate protein synthesis of their targeting mRNAs at the post-transcriptional level, usually by suppressing their target gene expression. The miRNAs dysregulated during HCV infection could directly or indirectly modulate HCV replication and/or induce liver diseases. Regulatory mechanisms of various miRNAs in HCV replication and pathogenesis have been characterized. Some dysregulated miRNAs have been considered as the biomarkers for the detection of HCV infection and/or HCV-related diseases. In this review, we intend to briefly summarize the identified miRNAs functioning at HCV replication and pathogenesis, focusing on the recent developments.

Strategies of Influenza A Virus to Ensure the Translation of Viral mRNAs.

Viruses are obligatorily intracellular pathogens. To generate progeny virus particles, influenza A viruses (IAVs) have to divert the cellular machinery to ensure sufficient translation of viral mRNAs. To this end, several strategies have been exploited by IAVs, such as host gene shutoff, suppression of host innate immune responses, and selective translation of viral mRNAs. Various IAV proteins are responsible for host gene shutoff, e.g., NS1, PA-X, and RdRp, through inhibition of cellular gene transcription, suppression of cellular RNA processing, degradation of cellular RNAs, and blockage of cellular mRNA export from the nucleus. Host shutoff should suppress the innate immune responses and also increase the translation of viral mRNAs indirectly due to the reduced competition from cellular mRNAs for cellular translational machinery. However, many other mechanisms are also responsible for the suppression of innate immune responses by IAV, such as prevention of the detection of the viral RNAs by the RLRs, inhibition of the activities of proteins involved in signaling events of interferon production, and inhibition of the activities of interferon-stimulated genes, mainly through viral NS1, PB1-F2, and PA-X proteins. IAV mRNAs may be selectively translated in favor of cellular mRNAs through interacting with viral and/or cellular proteins, such as NS1, PABPI, and/or IFIT2, in the 5'-UTR of viral mRNAs. This review briefly summarizes the strategies utilized by IAVs to ensure sufficient translation of viral mRNAs focusing on recent developments.

Cellular PSMB4 Protein Suppresses Influenza A Virus Replication through Targeting NS1 Protein.

The nonstructural protein 1 (NS1) of influenza A virus (IAV) possesses multiple functions, such as the inhibition of the host antiviral immune responses, to facilitate viral infection. To search for cellular proteins interacting with the IAV NS1 protein, the yeast two-hybrid system was adopted. Proteasome family member PSMB4 (proteasome subunit beta type 4) was found to interact with the NS1 protein in this screening experiment. The binding domains of these two proteins were also determined using this system. The physical interactions between the NS1 and cellular PSMB4 proteins were further confirmed by co-immunoprecipitation assay and confocal microscopy in mammalian cells. Neither transiently nor stably expressed NS1 protein affected the PSMB4 expression in cells. In contrast, PSMB4 reduced the NS1 protein expression level, especially in the presence of MG132. As expected, the functions of the NS1 protein, such as inhibition of interferon activity and enhancement of transient gene expression, were suppressed by PSMB4. PSMB4 knockdown enhances IAV replication, while its overexpression attenuates IAV replication. Thus, the results of this study suggest that the cellular PSMB4 protein interacts with and possibly facilitates the degradation of the NS1 protein, which in turn suppresses IAV replication.

Immuno-modulatory activity of Ganoderma lucidum-derived polysacharide on human monocytoid dendritic cells pulsed with Der p 1 allergen.

BACKGROUND: Ganoderma lucidum-derived polysaccharide (PS-G) can rapidly and effectively promote the activation and maturation of immature dendritic cells (DCs), suggesting that PS-G possesses the capacity to regulate immune responses. This study aimed to clarify the immunologic effect of PS-G on monocyte-derived dendritic cells (MD-DCs) from asthmatic children allergic to house dust mites. The MD-DCs were stimulated for 24 h with the related allergen, Der p 1, in the presence or absence of PS-G. Cell surface markers and phagocytic capacity were assessed by FACS analysis, and key polarizing cytokines (IL-12 p40, IL-12 p70, IL-6, IL-23, and IL-10) were quantified. The subsequent regulatory effect of pulsed MD-DCs on naïve T cells was evaluated by determining the T-cell cytokine profile. RESULTS: PS-G induced the maturation of MD-DCs and decreased phagocytic capacity, even if pulsed with Der p 1. After incubation with PS-G and Der p 1, MD-DCs produced higher amounts of IL-12 p70, IL-12 p40, IL-6, IL-23, and IL10 than Der p 1-pulsed DCs. Furthermore, type 1 helper T (Th1) cell cytokine (INF-γ) production was highly increased when naïve autologous T cells were co-cultured with Der p 1-pulsed MD-DCs. Naïve T cells stimulated by MD-DCs pulsed with Der p 1 failed to produce proliferation of T-cells, whereas the addition of PS-G to Der p 1 induced a significant proliferation of T-cells similar to that observed with PS-G alone. CONCLUSION: The presence of PS-G in an allergen pulse promoted allergic MD-DCs to produce IL-12 p70, IL-12 p40, IL-6, IL-23, and IL-10, and exerted an effect on shifting the immune balance towards Th1 in children with allergic asthma.

Visualization of the structures of the hepatitis C virus replication complex.

Hepatitis C viral RNA synthesis has been demonstrated to occur on a lipid raft membrane structure. Lipid raft membrane fraction purified by membrane flotation analysis was observed using transmission electron microscopy and atomic force microscopy. Particles around 0.7 um in size were found in lipid raft membrane fraction purified from hepatitis C virus (HCV) replicon but not their parental HuH7 cells. HCV NS5A protein was associated with these specialized particles. After several cycles of freezing-thawing, these particles would fuse into larger sizes up to 10 um. Knockdown of seven proteins associated with lipid raft (VAPA, COPG, RAB18, COMT, CDC42, DPP4, and KDELR2) of HCV replicon cells reduced the observed number of these particles and suppressed the HCV replication. Results in this study indicated that HCV replication complexes with associated lipid raft membrane form distinct particle structures of around 0.7 um as observed from transmission electron microscopy and atomic force microscopy.

Hepatitis C Viral Replication Complex.

The life cycle of the hepatitis C virus (HCV) can be divided into several stages, including viral entry, protein translation, RNA replication, viral assembly, and release. HCV genomic RNA replication occurs in the replication organelles (RO) and is tightly linked to ER membrane alterations containing replication complexes (proteins NS3 to NS5B). The amplification of HCV genomic RNA could be regulated by the RO biogenesis, the viral RNA structure (i.e., cis-acting replication elements), and both viral and cellular proteins. Studies on HCV replication have led to the development of direct-acting antivirals (DAAs) targeting the replication complex. This review article summarizes the viral and cellular factors involved in regulating HCV genomic RNA replication and the DAAs that inhibit HCV replication.

Cellular factors involved in the hepatitis C virus life cycle.

The hepatitis C virus (HCV), an obligatory intracellular pathogen, highly depends on its host cells to propagate successfully. The HCV life cycle can be simply divided into several stages including viral entry, protein translation, RNA replication, viral assembly and release. Hundreds of cellular factors involved in the HCV life cycle have been identified over more than thirty years of research. Characterization of these cellular factors has provided extensive insight into HCV replication strategies. Some of these cellular factors are targets for anti-HCV therapies. In this review, we summarize the well-characterized and recently identified cellular factors functioning at each stage of the HCV life cycle.

Antibodies Targeting Two Epitopes in SARS-CoV-2 Neutralize Pseudoviruses with the Spike Proteins from Different Variants.

The COVID-19 pandemic was caused by SARS-CoV-2 infection. To prevent the spread of SARS-CoV-2, an effective vaccine is required. Two linear peptides from potential B-cell epitopes in the spike protein of SARS-CoV-2 (a.a. 440-460; a.a. 494-506) were synthesized and used to immunize rabbits. High-titer antibodies of IgG were produced, purified, and verified by Western blot analysis. Antibodies against these two epitopes could effectively neutralize SARS-CoV-2 pseudoviral particles with the spike proteins from not only the original strain (basal; wild-type), but also a strain with a single point mutation (D614G), and two other emerging variants (the Alpha and Beta variants) prevalent around the world, but not from SARS-CoV. In conclusion, antibodies against these two epitopes are protective. This information is important for the development of vaccines against SARS-CoV-2.

Nanosized Particles Assembled by a Recombinant Virus Protein Are Able to Encapsulate Negatively Charged Molecules and Structured RNA.

RNA-based molecules have recently become hot candidates to be developed into therapeutic agents. However, successful applications of RNA-based therapeutics might require suitable carriers to protect the RNA from enzymatic degradation by ubiquitous RNases in vivo. Because of their better biocompatibility and biodegradability, protein-based nanoparticles are considered to be alternatives to their synthetic polymer-based counterparts for drug delivery. Hepatitis C virus (HCV) core protein has been suggested to be able to self-assemble into nucleocapsid-like particles in vitro. In this study, the genomic RNA-binding domain of HCV core protein consisting of 116 amino acids (p116) was overexpressed with E. coli for investigation. The recombinant p116 was able to assemble into particles with an average diameter of approximately 27 nm, as visualized by electron microscopy and atomic force microscopy. Measurements with fluorescence spectroscopy, flow cytometry, and fluorescence quenching indicated that the p116-assembled nanoparticles were able to encapsulate small anionic molecules and structured RNA. This study demonstrates methods that exploit the self-assembly nature of a virus-derived protein for nanoparticle production. This study also suggests that the virus-derived protein-assembled particles could possibly be developed into potential carriers for anionic molecular drugs and structured RNA-based therapeutics.

Enterovirus type 71 2A protease functions as a transcriptional activator in yeast.

Enterovirus type 71 (EV71) 2A protease exhibited strong transcriptional activity in yeast cells. The transcriptional activity of 2A protease was independent of its protease activity. EV71 2A protease retained its transcriptional activity after truncation of 40 amino acids at the N-terminus but lost this activity after truncation of 60 amino acids at the N-terminus or deletion of 20 amino acids at the C-terminus. Thus, the acidic domain at the C-terminus of this protein is essential for its transcriptional activity. Indeed, deletion of amino acids from 146 to 149 (EAME) in this acidic domain lost the transcriptional activity of EV71 2A protein though still retained its protease activity. EV71 2A protease was detected both in the cytoplasm and nucleus using confocal microscopy analysis. Coxsackie virus B3 2A protease also exhibited transcriptional activity in yeast cells. As expected, an acidic domain in the C-terminus of Coxsackie virus B3 2A protease was also identified. Truncation of this acidic domain resulted in the loss of transcriptional activity. Interestingly, this acidic region of poliovirus 2A protease is critical for viral RNA replication. The transcriptional activity of the EV71 or Coxsackie virus B3 2A protease should play a role in viral replication and/or pathogenesis.

MicroRNA-Independent Modulation of DICER1 Expression by hAgo2.

Many proteins, including DICER1 and hAgo2, are involved in the biogenesis of microRNAs (miRNAs). Whether hAgo2 regulates DICER1 expression is unknown. Exogenously overexpressed hAgo2 suppressed DICER1 expression at the levels of both protein and mRNA, and the reduction in hAgo2 expression enhanced DICER1 expression. Precursor miRNA processing mediated by DICER1 was also modulated by hAgo2. However, hAgo2 protein did not suppress DICER1 promoter activity. Therefore, hAgo2 protein probably regulates DICER1 expression at the posttranscriptional level. Indeed, hAgo2 protein inhibited the reporter assay of the DICER1 mRNA 3' untranslated region (3'-UTR). Previous reports have demonstrated that miRNAs (e.g., let-7 and miR-103/107) inhibited DICER1 expression posttranscriptionally. However, hAgo2 still suppressed DICER1 expression in the cells depleted of these miRNAs. Moreover, the reporter activities of the DICER1 mRNA 3'-UTR without these miRNA binding sites were still suppressed by hAgo2. Therefore, in addition to an miRNA-dependent pathway, hAgo2 can also modulate DICER1 expression through an miRNA-independent mechanism. Downregulation of DICER1 expression was further proven to be dependent on both hAgo2 and AUF1 proteins. Interactions of hAgo2 and AUF1 proteins were demonstrated by the coimmunoprecipitation assay. As expected, hAgo2 could not suppress the DICER1 mRNA 3'-UTR reporter with a mutation in the potential AUF1-binding site. Thus, downregulation of DICER1 expression through the 3'-UTR requires both hAgo2 and AUF1.

Increased ATP generation in the host cell is required for efficient vaccinia virus production.

To search for cellular genes up-regulated by vaccinia virus (VV) infection, differential display-reverse transcription-polymerase chain reaction (ddRT-PCR) assays were used to examine the expression of mRNAs from mock-infected and VV-infected HeLa cells. Two mitochondrial genes for proteins that are part of the electron transport chain that generates ATP, ND4 and CO II, were up-regulated after VV infection. Up-regulation of ND4 level by VV infection was confirmed by Western blotting analysis. Up-regulation of ND4 was reduced by the MAPK inhibitor, apigenin, which has been demonstrated elsewhere to inhibit VV replication. The induction of ND4 expression occurred after viral DNA replication since ara C, an inhibitor of poxviral DNA replication, could block this induction. ATP production was increased in the host cells after VV infection. Moreover, 4.5 microM oligomycin, an inhibitor of ATP production, reduced the ATP level 13 hr after virus infection to that of mock-infected cells and inhibited viral protein expression and virus production, suggesting that increased ATP production is required for efficient VV production. Our results further suggest that induction of ND4 expression is through a Bcl-2 independent pathway.

Expression and membrane integration of SARS-CoV E protein and its interaction with M protein.

The severe acute respiratory syndrome (SARS)-CoV E gene fragment was cloned and expressed as a recombinant protein fused with a myc tag at the N-terminus in vitro and in Vero E6 cells. Similar to other N-glycosylated proteins, the glycosylation of SARS-CoV E protein occurred co-translationally in the presence of microsomes. The SARS-CoV E protein is predicted to be a double-spanning membrane protein lacking a conventional signal peptide. Both of the transmembrane regions (a.a. 11-33 and 37-59) are predicted to be alpha-helices, which penetrate into membranes by themselves. As expected, these two transmembrane regions inserted a cytoplasmic protein into the endoplasmic reticulum membrane. Either of these two transmembrane domains co-localized with M protein. Both the transmembrane domains of E protein are required to interact with M protein, while either of the hydrophilic regions (a.a. 1-10 or 60-76) is dispensable as shown by co-immunoprecipitation assay. These results are important for the study of SARS-CoV assembly.

Interactions between M protein and other structural proteins of severe, acute respiratory syndrome-associated coronavirus.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) structural proteins (S, E, M, and NC) localize in different subcellular positions when expressed individually. However, SARS-CoV M protein is co-localized almost entirely with S, E, or NC protein when co-expressed in the cells. On the other hand, only partial co-localization was observed when S and E, S and NC, or E and NC were co-expressed in the cells. Interactions between SARS-CoV M and other structural proteins but not interactions between S and E, S and NC, or E and NC were further demonstrated by co-immunoprecipitation assay. These results indicate that SARS-CoV M protein, similar to the M proteins of other coronaviruses, plays a pivotal role in virus assembly. The cytoplasmic C-terminus domain of SARS-CoV M protein was responsible for binding to NC protein. Multiple regions of M protein interacted with E and S proteins. A model for the interactions between SARS-CoV M protein and other structural proteins is proposed. This study helps us better understand protein-protein interactions during viral assembly of SARS-CoV.