Alphavirus-based replicons demonstrate different interactions with host cells and can be optimized to increase protein expression.

IMPORTANCE: Alphavirus replicons are being developed as self-amplifying RNAs aimed at improving the efficacy of mRNA vaccines. These replicons are convenient for genetic manipulations and can express heterologous genetic information more efficiently and for a longer time than standard mRNAs. However, replicons mimic many aspects of viral replication in terms of induction of innate immune response, modification of cellular transcription and translation, and expression of nonstructural viral genes. Moreover, all replicons used in this study demonstrated expression of heterologous genes in cell- and replicon's origin-specific modes. Thus, many aspects of the interactions between replicons and the host remain insufficiently investigated, and further studies are needed to understand the biology of the replicons and their applicability for designing a new generation of mRNA vaccines. On the other hand, our data show that replicons are very flexible expression systems, and additional modifications may have strong positive impacts on protein expression.

Multiple Viral Protein Genome-Linked Proteins Compensate for Viral Translation in a Positive-Sense Single-Stranded RNA Virus Infection.

Viral protein genome-linked (VPg) protein plays an essential role in protein-primed replication of plus-stranded RNA viruses. VPg is covalently linked to the 5' end of the viral RNA genome via a phosphodiester bond typically at a conserved amino acid. Whereas most viruses have a single VPg, some viruses have multiple VPgs that are proposed to have redundant yet undefined roles in viral replication. Here, we use cricket paralysis virus (CrPV), a dicistrovirus that has four nonidentical copies of VPg, as a model to characterize the role of VPg copies in infection. Dicistroviruses contain two main open reading frames (ORFs) that are driven by distinct internal ribosome entry sites (IRESs). We systematically generated single and combinatorial deletions and mutations of VPg1 to VPg4 within the CrPV infectious clone and monitored viral yield in Drosophila S2 cells. Deletion of one to three VPg copies progressively decreased viral yield and delayed viral replication, suggesting a threshold number of VPgs for productive infection. Mass spectrometry analysis of CrPV VPg-linked RNAs revealed viral RNA linkage to either a serine or threonine in VPg, mutations of which in all VPgs attenuated infection. Mutating serine 4 in a single VPg abolished viral infection, indicating a dominant negative effect. Using viral minigenome reporters that monitor dicistrovirus 5' untranslated (UTR) and IRES translation revealed a relationship between VPg copy number and the ratio of distinct IRES translation activities. We uncovered a novel viral strategy whereby VPg copies in dicistrovirus genomes compensate for the relative IRES translation efficiencies to promote infection. IMPORTANCE Genetic duplication is exceedingly rare in small RNA viral genomes, as there is selective pressure to prevent RNA genomes from expanding. However, some small RNA viruses encode multiple copies of a viral protein, most notably an unusual viral protein that is linked to the viral RNA genome. Here, we investigate a family of viruses that contains multiple viral protein genome-linked proteins and reveal a novel viral strategy whereby viral protein copy number counterbalances differences in viral protein synthesis mechanisms.

Computational modeling of protracted HCMV replication using genome substrates and protein temporal profiles.

Human cytomegalovirus (HCMV) is a major cause of illness in immunocompromised individuals. The HCMV lytic cycle contributes to the clinical manifestations of infection. The lytic cycle occurs over ∼96 h in diverse cell types and consists of viral DNA (vDNA) genome replication and temporally distinct expression of hundreds of viral proteins. Given its complexity, understanding this elaborate system can be facilitated by the introduction of mechanistic computational modeling of temporal relationships. Therefore, we developed a multiplicity of infection (MOI)-dependent mechanistic computational model that simulates vDNA kinetics and late lytic replication based on in-house experimental data. The predictive capabilities were established by comparison to post hoc experimental data. Computational analysis of combinatorial regulatory mechanisms suggests increasing rates of protein degradation in association with increasing vDNA levels. The model framework also allows expansion to account for additional mechanisms regulating the processes. Simulating vDNA kinetics and the late lytic cycle for a wide range of MOIs yielded several unique observations. These include the presence of saturation behavior at high MOIs, inefficient replication at low MOIs, and a precise range of MOIs in which virus is maximized within a cell type, being 0.382 IU to 0.688 IU per fibroblast. The predicted saturation kinetics at high MOIs are likely related to the physical limitations of cellular machinery, while inefficient replication at low MOIs may indicate a minimum input material required to facilitate infection. In summary, we have developed and demonstrated the utility of a data-driven and expandable computational model simulating lytic HCMV infection.

Expression and purification of S5196-272 and S6200-317 proteins from Tilapia Lake Virus (TiLV) and their potential use as vaccines.

Tilapia Lake Virus Disease (TiLVD) is caused by Tilapia Lake Virus (TiLV), and it has a cumulative mortality rate of up to 90% in Nile tilapia (Oreochromis niloticus). TiLV is a negative enveloped single-stranded RNA virus with 10 genomic segments. Segment 5 (S5) and segment 6 (S6) were predicted to include a signaling peptide, suggesting that the encoded proteins of these two segments may exist as part of the virus envelope. Based on bioinformatic predictions, the S5 and S6 proteins in this study were produced, including S527-343, S527-172, S5196-272, S630-317, S630-190, and S6200-317. All proteins were tested for their expression in Escherichia coli. Only S5196-272 and S6200-317 were expressed as soluble and insoluble proteins, respectively. The soluble protein was purified using affinity chromatography, whereas the insoluble protein was solubilized using 6 M urea lysis buffer before purification. Both proteins were further purified using gel filtration chromatography, and the results showed a symmetric peak of both proteins suggested a high degree of uniformity in the conformation of these proteins. Antigenicity results indicated that these proteins were recognized by serum from TiLV-infected fish. The immunization tests revealed that serum antibodies levels in Nile tilapia produced by S5196-272 and S6200-317 were significantly increased (p-value < 0.05) at 7 days post-immunization (dpi) compared to antibody levels on Day 0 (D0). All the results combined suggested a potential vaccine candidate of S5 and S6 for TiLV protection in Nile tilapia.

Translation of Plant RNA Viruses.

Plant RNA viruses encode essential viral proteins that depend on the host translation machinery for their expression. However, genomic RNAs of most plant RNA viruses lack the classical characteristics of eukaryotic cellular mRNAs, such as mono-cistron, 5' cap structure, and 3' polyadenylation. To adapt and utilize the eukaryotic translation machinery, plant RNA viruses have evolved a variety of translation strategies such as cap-independent translation, translation recoding on initiation and termination sites, and post-translation processes. This review focuses on advances in cap-independent translation and translation recoding in plant viruses.

Development of Nanobodies against Mal de Río Cuarto virus major viroplasm protein P9-1 for diagnostic sandwich ELISA and immunodetection.

Mal de Río Cuarto virus (MRCV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in maize and is persistently and propagatively transmitted by planthopper vectors. Virus replication and assembly occur within viroplasms formed by viral and host proteins. This work describes the isolation and characterization of llama-derived Nanobodies (Nbs) recognizing the major viral viroplasm component, P9-1. Specific Nbs were selected against recombinant P9-1, with affinities in the nanomolar range as measured by surface plasmon resonance. Three selected Nbs were fused to alkaline phosphatase and eGFP to develop a sandwich ELISA test which showed a high diagnostic sensitivity (99.12%, 95% CI 95.21-99.98) and specificity (100%, 95% CI 96.31-100) and a detection limit of 0.236 ng/ml. Interestingly, these Nanobodies recognized different P9-1 conformations and were successfully employed to detect P9-1 in pull-down assays of infected maize extracts. Finally, we demonstrated that fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections. The Nbs developed in this work will aid the study of MRCV epidemiology, assist maize breeding programs, and be valuable tools to boost fundamental research on viroplasm structure and maturation.

Analysis of SARS-CoV-2 infection dynamic in vivo using reporter-expressing viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, is one of the biggest threats to public health. However, the dynamic of SARS-CoV-2 infection remains poorly understood. Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing recombinant (r)SARS-CoV-2 in the locus of the open reading frame (ORF)7a protein have jeopardized their use to monitor the dynamic of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the highly expressed viral nucleocapsid (N) gene followed by a porcine tescherovirus (PTV-1) 2A proteolytic cleavage site. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and excised lungs or whole organism of infected K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice. Importantly, real-time viral infection was readily tracked using a noninvasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2, in which a viral gene was not deleted, not only retained wild-type (WT) virus-like pathogenicity in vivo but also exhibited high stability in vitro and in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis, and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

Neural differentiation of glioblastoma cell lines via a herpes simplex virus thymidine kinase/ganciclovir system driven by a glial fibrillary acidic protein promoter.

Glioblastoma is a malignant brain tumor with poor prognosis that rapidly acquires resistance to available clinical treatments. The herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) system produces the selective elimination of HSVtk-positive cells and is a candidate for preclinical testing against glioblastoma via its ability to regulate proliferation and differentiation. Therefore, in this study, we aimed to establish a plasmid encoding the HSVtk/GCV system driven by a glial fibrillary acidic protein (GFAP) promoter and verify its possibility of neural differentiation of glioblastoma cell line under the GCV challenge. Four stable clones-N2A-pCMV-HSVtk, N2A-pGFAP-HSVtk, U251-pCMV-HSVtk, and U251-pGFAP-HSVtk-were established from neuronal N2A and glioblastoma U251 cell lines. In vitro GCV sensitivity was assessed by MTT assay for monitoring time- and dosage-dependent cytotoxicity. The capability for neural differentiation in stable glioblastoma clones during GCV treatment was assessed by performing immunocytochemistry for nestin, GFAP, and ßIII-tubulin. Under GFAP promoter control, the U251 stable clone exhibited GCV sensitivity, while the neuronal N2A clones were nonreactive. During GCV treatment, cells underwent apoptosis on day 3 and dying cells were identified after day 5. Nestin was increasingly expressed in surviving cells, indicating that the population of neural stem-like cells was enriched. Lower levels of GFAP expression were detected in surviving cells. Furthermore, ßIII-tubulin-positive neuron-like cells were identified after GCV treatment. This study established pGFAP-HSVtk-P2A-EGFP plasmids that successfully ablated GFAP-positive glioblastoma cells, but left neuronal N2A cells intact. These data suggest that the neural differentiation of glioblastoma cells can be promoted by treatment with the HSVtk/GCV system.

Complete genome sequence of a HPV31 isolate from laryngeal squamous cell carcinoma and biological consequences for p97 promoter activity.

Human papillomavirus type 31, although detected less frequently than HPV types 16 and 18, is associated with head and neck squamous cell carcinomas. Previous studies suggest that polymorphisms in the long control region (LCR) may alter the oncogenic potential of the virus. This study reports the first complete genome of a South African HPV31 isolate from a laryngeal squamous cell carcinoma. Sequence variations relative to the HPV31 prototype sequence were identified. The pBlue-Topo® vector, a reporter gene system was used to investigate the possible influence of these variations on the LCR promoter activity in vitro. Using mutagenesis to create two different fragments, ß-galactosidase assays were used to monitor the effect of nucleotide variations on the p97 promoter. Increased ß-galactosidase expression was observed in mutants when compared to the South African HPV31 LCR isolate. Enhanced transcriptional activity was observed with the mutant that possessed a single nucleotide change within the YY1 transcription factor binding site. In conclusion, sequence variation within the LCR of HPV31 isolates may have a functional effect on viral p97 promoter activity.

Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome.

Programmed ribosomal frameshifting is a key event during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome that allows synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here, we present the cryo-electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal messenger RNA (mRNA) channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel. Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, we compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.

FUS/TLS Suppresses Enterovirus Replication and Promotes Antiviral Innate Immune Responses.

During viral infection, the dynamic virus-host relationship is constantly in play. Many cellular proteins, such as RNA-binding proteins (RBPs), have been shown to mediate antiviral responses during viral infection. Here, we report that the RBP FUS/TLS (fused in sarcoma/translocated in liposarcoma) acts as a host-restricting factor against infection with coxsackievirus B3 (CVB3). Mechanistically, we found that deletion of FUS leads to increased viral RNA transcription and enhanced internal ribosome entry site (IRES)-driven translation, with no apparent impact on viral RNA stability. We further demonstrated that FUS physically interacts with the viral genome, which may contribute to direct inhibition of viral RNA transcription/translation. Moreover, we identified a novel function for FUS in regulating host innate immune response. We show that in the absence of FUS, gene expression of type I interferons and proinflammatory cytokines elicited by viral or bacterial infection is significantly impaired. Emerging evidence suggests a role for stress granules (SGs) in antiviral innate immunity. We further reveal that knockout of FUS abolishes the ability to form SGs upon CVB3 infection or poly(I·C) treatment. Finally, we show that, to avoid FUS-mediated antiviral response and innate immunity, CVB3 infection results in cytoplasmic mislocalization and cleavage of FUS through the enzymatic activity of viral proteases. Together, our findings in this study identify FUS as a novel host antiviral factor which restricts CVB3 replication through direct inhibition of viral RNA transcription and protein translation and through regulation of host antiviral innate immunity.IMPORTANCE Enteroviruses are common human pathogens, including those that cause myocarditis (coxsackievirus B3 [CVB3]), poliomyelitis (poliovirus), and hand, foot, and mouth disease (enterovirus 71). Understanding the virus-host interaction is crucial for developing means of treating and preventing diseases caused by these pathogens. In this study, we explored the interplay between the host RNA-binding protein FUS/TLS and CVB3 and found that FUS/TLS restricts CVB3 replication through direct inhibition of viral RNA transcription/translation and through regulation of cellular antiviral innate immunity. To impede the antiviral role of FUS, CVB3 targets FUS for mislocalization and cleavage. Findings from this study provide novel insights into interactions between CVB3 and FUS, which may lead to novel therapeutic interventions against enterovirus-induced diseases.

The antiviral effect of metformin on zika and dengue virus infection.

The Dengue (DENV) and zika (ZIKV) virus infections are currently a public health concern. At present, there is no treatment or a safe and effective vaccine for these viruses. Hence, the development of new strategies as host-directed therapy is required. In this sense, Metformin (MET), an FDA-approved drug used for the treatment of type 2 diabetes, has shown an anti-DENV effect in vitro by activating AMPK and reducing HMGCR activity. In this study, MET treatment was evaluated during in vitro and in vivo ZIKV infection and compared to MET treatment during DENV infection. Our results demonstrated that MET has a broad in vitro antiviral spectrum. MET inhibited ZIKV infection in different cell lines, but it was most effective in inhibiting DENV and yellow fever virus (YFV) infection in Huh-7 cells. However, the drug failed to protect against ZIKV infection when AG129 immunodeficient mice were used as in vivo model. Interestingly, MET increased DENV-infected male mice's survival time, reducing the severe signs of the disease. Together, these findings indicate that, although MET was an effective antiviral agent to inhibit in vitro and in vivo DENV infection, it could only inhibit in vitro ZIKV infection.

MRC5 cells engineered to express ACE2 serve as a model system for the discovery of antivirals targeting SARS-CoV-2.

Although the spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has resulted in a worldwide pandemic, there are currently no virus-specific drugs that are fully effective against SARS-CoV-2. Only a limited number of human-derived cells are capable of supporting SARS-CoV-2 replication and the infectivity of SARS-CoV-2 in these cells remains poor. In contrast, monkey-derived Vero cells are highly susceptibility to infection with SARS-CoV-2, although they are not suitable for the study of antiviral effects by small molecules due to their limited capacity to metabolize drugs compared to human-derived cells. In this study, our goal was to generate a virus-susceptible human cell line that would be useful for the identification and testing of candidate drugs. Towards this end, we stably transfected human lung-derived MRC5 cells with a lentiviral vector encoding angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2. Our results revealed that SARS-CoV-2 replicates efficiently in MRC5/ACE2 cells. Furthermore, viral RNA replication and progeny virus production were significantly reduced in response to administration of the replication inhibitor, remdesivir, in MRC5/ACE2 cells compared with Vero cells. We conclude that the MRC5/ACE2 cells will be important in developing specific anti-viral therapeutics and will assist in vaccine development to combat SARS-CoV-2 infections.

Human Schlafen 11 exploits codon preference discrimination to attenuate viral protein synthesis of prototype foamy virus (PFV).

Recently, the Schlafen (SLFN) proteins have been identified as a novel interferon-stimulated family with antiviral properties. In this study, we reported that SLFN11 inhibited prototype foamy virus (PFV) replication. Over-expression of human SLFN11 reduced viral production, while knockdown of SLFN11 enhanced viral infectivity. In addition, SLFN11 from cattle and African green monkey also suppressed PFV production. Both the ATPase activity and helicase activity of SLFN11 were required for its inhibitory function. Dephosphorylation activated the antiviral activity of SLFN11. More importantly, SLFN11 inhibited the expression of viral protein, which was rescued by viral gene codon optimization. Together, our results demonstrated that SLFN11 impaired PFV viral protein synthesis by exploiting the distinct codon usage between the virus and the host. These findings further broaden our understanding of the antiviral properties of the SLFN family and the molecular mechanism of PFV latent infection.

Kidney Subcapsular Allograft Transplants as a Model to Test Virus-Derived Chemokine-Modulating Proteins as Therapeutics.

Solid tissue transplant is a growing medical need that is further complicated by a limited donor organ supply. Acute and chronic rejection occurs in nearly all transplants and reduces long-term graft survival, thus increasing the need for repeat transplantation. Viruses have evolved highly adapted responses designed to evade the host's immune defenses. Immunomodulatory proteins derived from viruses represent a novel class of potential therapeutics that are under investigation as biologics to attenuate immune-mediated rejection and damage. These immune-modulating proteins have the potential to reduce the need for traditional posttransplant immune suppressants and improve graft survival. The myxoma virus-derived protein M-T7 is a promising biologic that targets chemokine and glycosaminoglycan pathways central to kidney transplant rejection. Orthotopic transplantations in mice are prohibitively difficult and costly and require a highly trained microsurgeon to successfully perform the procedure. Here we describe a kidney-to-kidney subcapsular transplant model as a practical and simple method for studying transplant rejection, a model that requires fewer mice. One kidney can be used as a donor for transplants into six or more recipient mice. Using this model there is lower morbidity, pain, and mortality for the mice. Subcapsular kidney transplantation provides a first step approach to testing virus-derived proteins as new potential immune-modulating therapeutics to reduce transplant rejection and inflammation.

A Mouse Model of Acute Liver Injury by Warm, Partial Ischemia-Reperfusion for Testing the Efficacy of Virus-Derived Therapeutics.

Ischemia-reperfusion injury (IRI) drives early and long-term damage to organs as well as compounding damage from acute transplant rejection and surgical trauma. IRI initiates an aggressive and prolonged inflammation leading to tissue injury, organ failure, and death. However, there are few effective therapeutic interventions for IRI. The destructive inflammatory cell activity in IRI is part of an aberrant innate immune response that triggers multiple pathways. Hence, immune-modulating treatments to control pathways triggered by IRI hold great therapeutic potential. Viruses, especially large DNA viruses, have evolved highly effective immune-modulating proteins for the purpose of immune evasion and to protect the virus from the host immune defenses. A number of these immune-modulating proteins have proven therapeutically effective in preclinical models, many with function targeting pathways known to be involved in IRI. The use of virus-derived immune-modulating proteins thus represents a promising source for new treatments to target ischemia-reperfusion injury. Laboratory small animal models of IRI are well established and are able to reproduce many aspects of ischemia-reperfusion injury seen in humans. This chapter will discuss the methods used to perform the IRI procedure in mice, as well as clinically relevant diagnostic tests to evaluate liver injury and approaches for assessing histological damage while testing novel immune modulating protein treatments.

The emerging SARS-CoV-2 papain-like protease: Its relationship with recent coronavirus epidemics.

The papain-like protease (PLpro ) is an important enzyme for coronavirus polyprotein processing, as well as for virus-host immune suppression. Previous studies reveal that a molecular analysis of PLpro indicates the catalytic activity of viral PLpro and its interactions with ubiquitin. By using sequence comparisons, molecular models, and protein-protein interaction maps, PLpro was compared in the three recorded fatal CoV epidemics, which involved severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome CoV (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV). The pairwise sequence comparison of SARS-CoV-2 PLpro indicated similarity percentages of 82.59% and 30.06% with SARS-CoV PLpro and MERS-CoV PLpro , respectively. In comparison with SARS-CoV PLpro , in SARS-CoV-2, the PLpro had a conserved catalytic triad of C111, H278, and D293, with a slightly lower number of polar interface residues and of hydrogen bonds, a higher number of buried interface sizes, and a lower number of residues that interact with ubiquitin and PLpro . These features might contribute to a similar or slightly lower level of deubiquitinating activity in SARS-CoV-2 PLpro. It was, however, a much higher level compared to MERS-CoV, which contained amino acid mutations and a low number of polar interfaces. SARS-CoV-2 PLpro and SARS-CoV PLpro showed almost the same catalytic site profiles, interface area compositions and polarities, suggesting a general similarity in deubiquitination activity. Compared with MERS-CoV, SARS-CoV-2 had a higher potential for binding interactions with ubiquitin. These estimated parameters contribute to the knowledge gap in understanding how the new virus interacts with the immune system.

The coding capacity of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic1. To understand the pathogenicity and antigenic potential of SARS-CoV-2 and to develop therapeutic tools, it is essential to profile the full repertoire of its expressed proteins. The current map of SARS-CoV-2 coding capacity is based on computational predictions and relies on homology with other coronaviruses. As the protein complement varies among coronaviruses, especially in regard to the variety of accessory proteins, it is crucial to characterize the specific range of SARS-CoV-2 proteins in an unbiased and open-ended manner. Here, using a suite of ribosome-profiling techniques2-4, we present a high-resolution map of coding regions in the SARS-CoV-2 genome, which enables us to accurately quantify the expression of canonical viral open reading frames (ORFs) and to identify 23 unannotated viral ORFs. These ORFs include upstream ORFs that are likely to have a regulatory role, several in-frame internal ORFs within existing ORFs, resulting in N-terminally truncated products, as well as internal out-of-frame ORFs, which generate novel polypeptides. We further show that viral mRNAs are not translated more efficiently than host mRNAs; instead, virus translation dominates host translation because of the high levels of viral transcripts. Our work provides a resource that will form the basis of future functional studies.

Downregulation of T7 RNA polymerase transcription enhances pET-based recombinant protein production in Escherichia coli BL21 (DE3) by suppressing autolysis.

Escherichia coli BL21 (DE3) is an excellent and widely used host for recombinant protein production. Many variant hosts were developed from BL21 (DE3), but improving the expression of specific proteins remains a major challenge in biotechnology. In this study, we found that when BL21 (DE3) overexpressed glucose dehydrogenase (GDH), a significant industrial enzyme, severe cell autolysis was induced. Subsequently, we observed this phenomenon in the expression of 10 other recombinant proteins. This precludes a further increase of the produced enzyme activity by extending the fermentation time, which is not conducive to the reduction of industrial enzyme production costs. Analysis of membrane structure and messenger RNA expression analysis showed that cells could underwent a form of programmed cell death (PCD) during the autolysis period. However, blocking three known PCD pathways in BL21 (DE3) did not completely alleviate autolysis completely. Consequently, we attempted to develop a strong expression host resistant to autolysis by controlling the speed of recombinant protein expression. To find a more suitable protein expression rate, the high- and low-strength promoter lacUV5 and lac were shuffled and recombined to yield the promoter variants lacUV5-1A and lac-1G. The results showed that only one base in lac promoter needs to be changed, and the A at the +1 position was changed to a G, resulting in the improved host BL21 (DE3-lac1G), which resistant to autolysis. As a consequence, the GDH activity at 43 h was greatly increased from 37.5 to 452.0 U/ml. In scale-up fermentation, the new host was able to produce the model enzyme with a high rate of 89.55 U/ml/h at 43 h, compared to only 3 U/ml/h achieved using BL21 (DE3). Importantly, BL21 (DE3-lac1G) also successfully improved the production of 10 other enzymes. The engineered E. coli strain constructed in this study conveniently optimizes recombinant protein overexpression by suppressing cell autolysis, and shows great potential for industrial applications.

Cloning, expression and characterization of P1 and NIa proteases from banana bract mosaic virus (BBrMV).

Banana bract mosaic virus (BBrMV) causes the banana bract mosaic disease in banana. It belongs to the genus Potyvirus within the family Potyviridae. To the best of our knowledge apart from BBrMV coat protein gene, there are no reports on cloning, expression and characterization of any other genes from BBrMV. In this study, the BBrMV P1 and NIa protease genes were amplified from BBrMV infected banana plant cultivar Nendran and were cloned into the protein expression vector pET28b. Recombinant plasmids were transferred to BL21-CodonPlus (DE3)-RP cells and the IPTG (Isopropyl ß-d-1-thiogalactopyranoside) induced BBrMV P1 and NIa proteins with molecular weights of 42 and 32 KDa respectively were purified on Ni-NTA resin column under denaturing conditions using 8 M urea. BBrMV P1 and NIa purified proteins were detected by Western blot using anti-histidine antibody. The activity of both P1 and NIa proteases in native form was analyzed through in-gel zymographic assay. The activities of both the proteases were strongly inhibited by PMSF, suggesting that both the proteases are the serine type proteases. Interestingly both the proteases showed a temperature optimum of 50 °C while the pH optimum was 8. Both proteases lost their activity when incubated at 70 °C for 1 h. This is the first report of expression, purification and characterization of BBrMV P1 and NIa proteases.