Enhancing recombinant antibody yield in Chinese hamster ovary cells.

A range of recombinant monoclonal antibodies (rMAbs) have found application in treating diverse diseases, spanning various cancers and immune system disorders. Chinese hamster ovary (CHO) cells have emerged as the predominant choice for producing these rMAbs due to their robustness, ease of transfection, and capacity for posttranslational modifications akin to those in human cells. Transient transfection and/or stable expression could be conducted to express rMAbs in CHO cells. To bolster the yield of rMAbs in CHO cells, a multitude of approaches have been developed, encompassing vector optimization, medium formulation, cultivation parameters, and cell engineering. This review succinctly outlines these methodologies when also addressing challenges encountered in the production process, such as issues with aggregation and fucosylation.

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.

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.

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.

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.

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.

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.

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.

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.

Toll-Like Receptor 3 Is Involved in Detection of Enterovirus A71 Infection and Targeted by Viral 2A Protease.

Enterovirus A71 (EV-A71) has emerged as a major pathogen causing hand, foot, and mouth disease, as well as neurological disorders. The host immune response affects the outcomes of EV-A71 infection, leading to either resolution or disease progression. However, the mechanisms of how the mammalian innate immune system detects EV-A71 infection to elicit antiviral immunity remain elusive. Here, we report that the Toll-like receptor 3 (TLR3) is a key viral RNA sensor for sensing EV-A71 infection to trigger antiviral immunity. Expression of TLR3 in HEK293 cells enabled the cells to sense EV-A71 infection, leading to type I, IFN-mediated antiviral immunity. Viral double-stranded RNA derived from EV-A71 infection was a key ligand for TLR3 detection. Silencing of TLR3 in mouse and human primary immune cells impaired the activation of IFN-ß upon EV-A71 infection, thus reinforcing the importance of the TLR3 pathway in defending against EV-A71 infection. Our results further demonstrated that TLR3 was a target of EV-A71 infection. EV-A71 protease 2A was implicated in the downregulation of TLR3. Together, our results not only demonstrate the importance of the TLR3 pathway in response to EV-A71 infection, but also reveal the involvement of EV-A71 protease 2A in subverting TLR3-mediated antiviral defenses.

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.

The Primary Mechanism of Cellular Internalization for a Short Cell- Penetrating Peptide as a Nano-Scale Delivery System.

BACKGROUND: Development of effective drug delivery systems (DDS) is a critical issue in health care and medicine. Advances in molecular biology and nanotechnology have allowed the introduction of nanomaterial-based drug delivery systems. Cell-penetrating peptides (CPPs) can form the basis of drug delivery systems by virtue of their ability to support the transport of cargoes into the cell. Potential cargoes include proteins, DNA, RNA, liposomes, and nanomaterials. These cargoes generally retain their bioactivities upon entering cells. METHOD: In the present study, the smallest, fully-active lactoferricin-derived CPP, L5a is used to demonstrate the primary contributor of cellular internalization. RESULTS: The secondary helical structure of L5a encompasses symmetrical positive charges around the periphery. The contributions of cell-specificity, peptide length, concentration, zeta potential, particle size, and spatial structure of the peptides were examined, but only zeta potential and spatial structure affected protein transduction efficiency. FITC-labeled L5a appeared to enter cells via direct membrane translocation insofar as endocytic modulators did not block FITC-L5a entry. This is the same mechanism of protein transduction active in Cy5 labeled DNA delivery mediated by FITC-L5a. A significant reduction of transduction efficiency was observed with structurally incomplete FITC-L5a formed by tryptic destruction, in which case the mechanism of internalization switched to a classical energydependent endocytosis pathway. CONCLUSION: These results support the continued development of the non-cytotoxic L5a as an efficient tool for drug delivery.

A Sequence in the loop domain of hepatitis C virus E2 protein identified in silico as crucial for the selective binding to human CD81.

Hepatitis C virus (HCV) is a species-specific pathogenic virus that infects only humans and chimpanzees. Previous studies have indicated that interactions between the HCV E2 protein and CD81 on host cells are required for HCV infection. To determine the crucial factors for species-specific interactions at the molecular level, this study employed in silico molecular docking involving molecular dynamic simulations of the binding of HCV E2 onto human and rat CD81s. In vitro experiments including surface plasmon resonance measurements and cellular binding assays were applied for simple validations of the in silico results. The in silico studies identified two binding regions on the HCV E2 loop domain, namely E2-site1 and E2-site2, as being crucial for the interactions with CD81s, with the E2-site2 as the determinant factor for human-specific binding. Free energy calculations indicated that the E2/CD81 binding process might follow a two-step model involving (i) the electrostatic interaction-driven initial binding of human-specific E2-site2, followed by (ii) changes in the E2 orientation to facilitate the hydrophobic and van der Waals interaction-driven binding of E2-site1. The sequence of the human-specific, stronger-binding E2-site2 could serve as a candidate template for the future development of HCV-inhibiting peptide drugs.

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.

Hepatitis C virus: Virology, diagnosis and treatment.

More than twenty years of study has provided a better understanding of hepatitis C virus (HCV) life cycle, including the general properties of viral RNA and proteins. This effort facilitates the development of sensitive diagnostic tools and effective antiviral treatments. At present, serologic screening test is recommended to perform on individuals in the high risk groups and nucleic acid tests are recommended to confirm the active HCV infections. Quantization and genotyping of HCV RNAs are important to determine the optimal duration of anti-viral therapy and predict the likelihood of response. In the early 2000s, pegylated interferon plus ribavirin became the standard anti-HCV treatment. However, this therapy is not ideal. To 2014, boceprevir, telaprevir, simeprevir, sofosbuvir and Harvoni are approved by Food and Drug Administration for the treat of HCV infections. It is likely that the new all-oral, interferon-free, pan-genotyping anti-HCV therapy will be available within the next few years. Majority of HCV infections will be cured by these anti-viral treatments. However, not all patients are expected to be cured due to viral resistance and the high cost of antiviral treatments. Thus, an efficient prophylactic vaccine will be the next challenge in the fight against HCV infection.

Cleavage of Dicer protein by I7 protease during vaccinia virus infection.

Dicer is the key component in the miRNA pathway. Degradation of Dicer protein is facilitated during vaccinia virus (VV) infection. A C-terminal cleaved product of Dicer protein was detected in the presence of MG132 during VV infection. Thus, it is possible that Dicer protein is cleaved by a viral protease followed by proteasome degradation of the cleaved product. There is a potential I7 protease cleavage site in the C-terminus of Dicer protein. Indeed, reduction of Dicer protein was detected when Dicer was co-expressed with I7 protease but not with an I7 protease mutant protein lack of the protease activity. Mutation of the potential I7 cleavage site in the C-terminus of Dicer protein resisted its degradation during VV infection. Furthermore, Dicer protein was reduced dramatically by recombinant VV vI7Li after the induction of I7 protease. If VV could facilitate the degradation of Dicer protein, the process of miRNA should be affected by VV infection. Indeed, accumulation of precursor miR122 was detected after VV infection or I7 protease expression. Reduction of miR122 would result in the suppression of HCV sub-genomic RNA replication, and, in turn, the amount of viral proteins. As expected, significant reduction of HCVNS5A protein was detected after VV infection and I7 protease expression. Therefore, our results suggest that VV could cleave Dicer protein through I7 protease to facilitate Dicer degradation, and in turn, suppress the processing of miRNAs. Effect of Dicer protein on VV replication was also studied. Exogenous expression of Dicer protein suppresses VV replication slightly while knockdown of Dicer protein does not affect VV replication significantly.

Studying coronavirus-host protein interactions.

To understand the molecular mechanisms of viral replication and pathogenesis, it is necessary to establish the virus-host protein interaction networks. The yeast two-hybrid system is a powerful proteomic approach to study protein-protein interactions. After the identification of specific cellular factors interacting with the target viral protein using the yeast two-hybrid screening system, co-immunoprecipitation and confocal microscopy analyses are often used to verify the virus-host protein interactions in cells. Identification of the cellular factors required for viral survival or eliminating virus infected cells could help scientists develop more effective antiviral drugs. Here we summarize a standard protocol used in our lab to study the coronavirus-host protein interactions, including yeast two-hybrid screening, co-immunoprecipitation, and immunofluorescence microscopy analyses.

Production and pathogenicity of hepatitis C virus core gene products.

Hepatitis C virus (HCV) is a major cause of chronic liver diseases, including steatosis, cirrhosis and hepatocellular carcinoma, and its infection is also associated with insulin resistance and type 2 diabetes mellitus. HCV, belonging to the Flaviviridae family, is a small enveloped virus whose positive-stranded RNA genome encoding a polyprotein. The HCV core protein is cleaved first at residue 191 by the host signal peptidase and further cleaved by the host signal peptide peptidase at about residue 177 to generate the mature core protein (a.a. 1-177) and the cleaved peptide (a.a. 178-191). Core protein could induce insulin resistance, steatosis and even hepatocellular carcinoma through various mechanisms. The peptide (a.a. 178-191) may play a role in the immune response. The polymorphism of this peptide is associated with the cellular lipid drop accumulation, contributing to steatosis development. In addition to the conventional open reading frame (ORF), in the +1 frame, an ORF overlaps with the core protein-coding sequence and encodes the alternative reading frame proteins (ARFP or core+1). ARFP/core+1/F protein could enhance hepatocyte growth and may regulate iron metabolism. In this review, we briefly summarized the current knowledge regarding the production of different core gene products and their roles in viral pathogenesis.