SARS-CoV-2 nucleocapsid protein, rather than spike protein, triggers a cytokine storm originating from lung epithelial cells in patients with COVID-19.

PURPOSE: The aim of this study was to elucidate the factors associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that may initiate cytokine cascades and correlate the clinical characteristics of patients with coronavirus disease 2019 (COVID-19) with their serum cytokine profiles. METHODS: Recombinant baculoviruses displaying SARS-CoV-2 spike or nucleocapsid protein were constructed and transfected into A549 cells and THP-1-derived macrophages, to determine which protein initiate cytokine release. SARS-CoV-2-specific antibody titers and cytokine profiles of patients with COVID-19 were determined, and the results were associated with their clinical characteristics, such as development of pneumonia or length of hospital stay. RESULTS: The SARS-CoV-2 nucleocapsid protein, rather than the spike protein, triggers lung epithelial A549 cells to express IP-10, RANTES, IL-16, MIP-1α, basic FGF, eotaxin, IL-15, PDGF-BB, TRAIL, VEGF-A, and IL-5. Additionally, serum CTACK, basic FGF, GRO-α, IL-1α, IL-1RA, IL-2Rα, IL-9, IL-15, IL-16, IL-18, IP-10, M-CSF, MIF, MIG, RANTES, SCGF-ß, SDF-1α, TNF-α, TNF-ß, VEGF, PDGF-BB, TRAIL, ß-NGF, eotaxin, GM-CSF, IFN-α2, INF-γ, and MCP-1 levels were considerably increased in patients with COVID-19. Among them, patients with pneumonia had higher serum IP-10 and M-CSF levels than patients without. Patients requiring less than 3 weeks to show negative COVID-19 tests after contracting COVID-19 had higher serum IP-10 levels than the remaining patients. CONCLUSION: Our study revealed that nucleocapsid protein, lung epithelial cells, and IP-10 may be potential targets for the development of new strategies to prevent, or control, severe COVID-19.

Establishment of a Pseudovirus Platform for Neuraminidase Inhibiting Antibody Analysis.

Neuraminidase (NA)-based immunity to influenza can be useful for protecting against novel antigenic variants. To develop safe and effective tools to assess NA-based immunity, we generated a baculovirus-based pseudotyped virus, N1-Bac, that expresses the full-length NA of the influenza A/California/07/2009 (H1N1)pdm09 strain. We evaluated the level of NA-inhibiting (NI) antibodies in the paired blood sera of influenza patients by means of an enzyme-linked lectin assay (ELLA) using the influenza virus or N1-Bac. Additionally, we evaluated the level of NA antibodies by means of the enzyme-linked immunosorbent assay (ELISA) with an N1-expressing Sf21 culture. We detected a strong correlation between our results from using the influenza virus and NA-Bac pseudoviruses to detect NI antibodies and a medium-strong correlation between NI antibodies and NA antibodies determined by an N1-cell ELISA, indicating that baculovirus-based platforms can be successfully used to evaluate NI or NA antibodies. Furthermore, animal experiments showed that immunization with N1-Bac protected against infection with a drift variant of the A/H1N1pdm09 influenza virus. Our results demonstrate that recombinant baculovirus can be an effective influenza pseudotype to evaluate influenza serologic immunity and protect against influenza virus infection.

Antigenic Characterization of Neuraminidase of Influenza A/H7N9 Viruses Isolated in Different Years.

Influenza outbreaks caused by A/H7N9 viruses have occurred since 2013. After 2016, A/H7N9 influenza viruses underwent evolutionary changes. In this study, we examined the antigenic properties of influenza neuraminidase (NA) of A/H7N9 viruses as part of a live influenza vaccine (LAIV). It was shown that neuraminidase inhibiting (NI) antibodies obtained after A/Anhui/1/2013(H7N9)-based LAIV vaccination did not inhibit A/Hong Kong/125/2017(H7N9) NA and vice versa. The A/Hong Kong/125/2017(H7N9)-based LAIV elicited higher levels of NI antibodies compared to the A/Anhui/1/2013(H7N9)-based LAIV after two doses. Thelow degree of coincidence of the antibody response to hemagglutinin (HA) and NA after LAIV vaccination allows us to consider an enzyme-linked lectin assay (ELLA) as an additional measure for assessing the immunogenicity of influenza vaccines. In mice, N9-reactive monoclonal antibodies (mABs) for the A/environment/Shanghai/RL01/2013(H7N9) influenza virus partially protected against lung infection from the A/Guangdong/17SF003/2016 IDCDC-RG56N(H7N9) virus, thus showing the cross-protective properties of monoclonal antibodies against the drift variant.

Corrigendum: Development of a Scrub Typhus Diagnostic Platform Incorporating Cell-Surface Display Technology.

[This corrects the article DOI: 10.3389/fimmu.2021.761136.].

Development of a Scrub Typhus Diagnostic Platform Incorporating Cell-Surface Display Technology.

Scrub typhus (ST), also known as tsutsugamushi disease and caused by rickettsia Orientia tsutsugamushi, is an underestimated fatal epidemic in the Asia-Pacific region, resulting in a million human infections each year. ST is easily misdiagnosed as clinical diagnosis is based on non-specific skin eschar and flu-like symptoms. Thus, the lack of accurate, convenient, and low-cost detection methods for ST poses a global health threat. To address this problem, we adopted baculovirus surface-display technology to express three variants of TSA56, the major membrane antigen of O. tsutsugamushi, as well as the passenger domain of ScaC (ScaC-PD), on insect Sf21 cell surfaces rather than biosafety level 3 bacteria in an enzyme-linked immunosorbent assay (ELISA). Recombinant TSA56 and ScaC-PD were all properly expressed and displayed on Sf21 cells. Our cell-based ELISA comprising the four antigen-displaying cell types interacted with monoclonal antibodies as well as serum samples from ST-positive field-caught rats. This cell-based ELISA presented high accuracy (96.3%), sensitivity (98.6%), and specificity (84.6%) when tested against the ST-positive rat sera. Results of a pilot study using human sera were also highly consistent with the results of immunofluorescence analyses. By adopting this approach, we circumvented complex purification and refolding processes required to generate recombinant O. tsutsugamushi antigens and reduced the need for expensive equipment and extensively trained operators. Thus, our system has the potential to become a widely used serological platform for diagnosing ST.

Vaccinia virus-based vaccines confer protective immunity against SARS-CoV-2 virus in Syrian hamsters.

COVID-19 in humans is caused by Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that belongs to the beta family of coronaviruses. SARS-CoV-2 causes severe respiratory illness in 10-15% of infected individuals and mortality in 2-3%. Vaccines are urgently needed to prevent infection and to contain viral spread. Although several mRNA- and adenovirus-based vaccines are highly effective, their dependence on the "cold chain" transportation makes global vaccination a difficult task. In this context, a stable lyophilized vaccine may present certain advantages. Accordingly, establishing additional vaccine platforms remains vital to tackle SARS-CoV-2 and any future variants that may arise. Vaccinia virus (VACV) has been used to eradicate smallpox disease, and several attenuated viral strains with enhanced safety for human applications have been developed. We have generated two candidate SARS-CoV-2 vaccines based on two vaccinia viral strains, MVA and v-NY, that express full-length SARS-CoV-2 spike protein. Whereas MVA is growth-restricted in mammalian cells, the v-NY strain is replication-competent. We demonstrate that both candidate recombinant vaccines induce high titers of neutralizing antibodies in C57BL/6 mice vaccinated according to prime-boost regimens. Furthermore, our vaccination regimens generated TH1-biased immune responses in mice. Most importantly, prime-boost vaccination of a Syrian hamster infection model with MVA-S and v-NY-S protected the hamsters against SARS-CoV-2 infection, supporting that these two vaccines are promising candidates for future development. Finally, our vaccination regimens generated neutralizing antibodies that partially cross-neutralized SARS-CoV-2 variants of concern.

Comparison of Chicken Immune Responses after Inoculation with H5 Avian Influenza Virus-like Particles Produced by Insect Cells or Pupae.

INTRODUCTION: Novel clade 2.3.4.4 H5 highly pathogenic avian influenza virus (HPAIV) outbreaks have occurred since early 2015 in Taiwan and impacted the island economically, like they have many countries. This research investigates the immunogenicity of two HPAIV-like particles to assess their promise as vaccine candidates. MATERIAL AND METHODS: The haemagglutinin (HA) gene derived from clade 2.3.4.4 H5 HPAIV and matrix protein 1 (M1) gene were cloned into the pFastBac Dual baculovirus vector. The resulting recombinant viruses were expressed in Spodoptera frugiperda moth (Sf)21 cells and silkworm pupae to generate Sf21 virus-like particles (VLP) and silkworm pupa VLP. Two-week-old specific pathogen-free chickens were immunised and their humoral and cellular immune responses were analysed. RESULTS: The silkworm pupa VLP had higher haemagglutination competence. Both VLP types elicited haemagglutination inhibition antibodies, anti-HA antibodies, splenic interferon gamma (IFN-γ) and interleukin 4 (IL-4) mRNA expression, and CD4+/CD8+ ratio elevation. However, chickens receiving silkworm pupa VLP exhibited a significantly higher anti-HA antibody titre in ELISA after vaccination. Although Sf21 VLP recipients expressed more IFN-γ and IL-4, the increase in IFN-γ did not significantly raise the CD4+/CD8+ ratio and the increase in IL-4 did not promote anti-HA antibodies. CONCLUSION: Both VLP systems possess desirable immunogenicity in vivo. However, in respect of immunogenic efficacy and the production cost, pupa VLP may be the superior vaccine candidate against clade 2.3.4.4 H5 HPAIV infection.

Identification and Quantification of Anti-Gp.Mur Antibodies in Human Serum Using an Insect-Cell-Based System.

Gp.Mur is a clinically relevant antigen of the MNS blood group system that is highly prevalent in several Asian populations. Its corresponding antibody, anti-Gp.Mur, has been implicated in hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Currently, identifying and confirming anti-Gp.Mur antibody presence in sera via agglutination of a panel of red blood cells (RBCs) is inefficient and difficult to quantify. Using a baculovirus expression system to express Gp.Mur antigen on insect cell surfaces, we have developed a quantitative cell-based system to confirm the presence of anti-Gp.Mur antibody in human serum. We obtained 10 serum samples preidentified as having anti-Gp.Mur antibody and another 4 samples containing noncorresponding antibodies from hospital patients. Insect cells displaying Gp.Mur antigen successfully adsorbed anti-Gp.Mur antibody in the sera and inhibited the RBC agglutination mediated by this antibody. By varying the concentration of Gp.Mur-displaying cells, we could grade levels of RBC agglutination by anti-Gp.Mur antibody. Densitometric analysis further enabled quantitative determinations of hemagglutination inhibition by Gp.Mur-displaying cells. We believe that this cell-based hemagglutination inhibition system greatly improves or supplements existing technology and is a convenient means for accurately identifying and quantifying anti-Gp.Mur antibody.

PEDV Infection Generates Conformation-Specific Antibodies That Can Be Effectively Detected by a Cell-Based ELISA.

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes serious and highly contagious enteric disease in swine worldwide. In this study, we constructed a recombinant baculovirus (S-Bac) expressing full-length spike protein of the virulent epidemic genotype 2b (G2b) PEDV strain for serological studies of infected pigs. We found that most spike-specific antibodies produced upon PEDV infection in pigs are conformation-specific and they could be detected on S-Bac-infected insect cells by immunofluorescent assay, but they were insensitive to Western blot analysis, the typical method for antiserum analysis. These results indicated that spike conformation is crucial for serum recognition. Since it is difficult to purify trimeric spike membrane protein for conventional enzyme-linked immunosorbent assay (ELISA), we used S-Bac to generate a novel cell-based ELISA for convenient PEDV detection. We analyzed 100 pig serum samples, and our cell-based ELISA exhibited a sensitivity of 100%, a specificity of 97%, and almost perfect agreement [Cohen's kappa coefficient value (κ) = 0.98] with immunocytochemical staining results. Our cell-based ELISA rapidly presented antigen for proper detection of conformation-specific antibodies, making PEDV detection more convenient, and it will be useful for detecting many viral diseases in the future.

An Integrated Platform for Serological Detection and Vaccination of COVID-19.

Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is an ongoing pandemic. Detection and vaccination are essential for disease control, but they are distinct and complex operations that require significant improvements. Here, we developed an integrated detection and vaccination system to greatly simplify these efforts. We constructed recombinant baculoviruses to separately display the nucleocapsid (N) and spike (S) proteins of SARS-CoV-2. Insect cells infected by the recombinant baculoviruses were used to generate a cell-based system to accurately detect patient serum. Notably, although well-recognized by our newly developed detection system in which S-displaying insect cells acted as antigen, anti-S antibodies from many patients were barely detectable by Western blot, evidencing that COVID-19 patients primarily produce conformation-dependent anti-S antibodies. Furthermore, the same baculovirus constructs can display N (N-Bac) or S (S-Bac) on the baculovirus envelope and serve as vector vaccines. Animal experiments show that S-Bac or N-Bac immunization in mice elicited a strong and specific antibody response, and S-Bac in particular stimulated effective neutralizing antibodies without the need for adjuvant. Our integrated system maintains antigen conformation and membrane structure to facilitate serum detection and antibody stimulation. Thus, compared with currently available technologies, our system represents a simplified and efficient platform for better SARS-CoV-2 detection and vaccination.

Oral administration of porcine epidemic diarrhea virus spike protein expressing in silkworm pupae failed to elicit immune responses in pigs.

The silkworm (Bombyx mori) and its pupae have been used for decades as nutritional additives and applied on the production of high-quality recombinant proteins via the baculovirus expression vector (BEV) system. The bio-capsule, the fat-rich body, and some body components of the silkworm pupae, which deliver antigens passing through the harsh environment of digestive tract and reaching the intestine, have been used as a vehicle for oral vaccines. In the present study, to develop a novel oral vaccine against porcine epidemic diarrhea virus (PEDV), the PEDV spike (S) protein was expressed in silkworm pupae and BmN cells using the BEV system. After three doses of oral administrations with 2-week intervals in pigs, neither PEDV S protein-specific humoral nor mucosal immune responses can be detected. The failure of eliciting the PEDV-specific immune response suggested that the BEV system using BmN cells or silkworm pupae as oral immunogen-expression vehicles was not able to overcome the immunological unresponsiveness, which was possibly due to gastrointestinal specific barriers and oral tolerance. Better strategies to enhance the delivery and immunogenicity of oral vaccines should be further investigated. Nevertheless, the PEDV S protein generated in the BmN cells and silkworm pupae herein provides an efficient tool to produce the recombinant antigen for future applications.

Baculovirus as Versatile Vectors for Protein Display and Biotechnological Applications.

The baculovirus-insect cell system has long been deployed for a variety of applications including for use as biopesticides, for recombinant protein production, transient transgene expression, tissue therapy, and for vaccine production. Apart from the advantage of large-scale heterologous protein production with appropriate eukaryotic post-translational modification, foreign proteins can also be displayed on the viral envelope. This surface-display technology preserves the native multimeric structure of the protein, thereby expanding the clinical and pharmaceutical utility of the baculovirus system. Recombinant baculoviruses displaying major antigens for human or animal viruses can serve as appropriate vaccines. They can also serve as effective diagnostic platforms and various cell-based assay systems. In this review, we discuss progress in applying baculovirus surface-display, including protein display on the envelope, capsid, and occlusion bodies of baculoviruses, as well as on cells. We will also describe strategies for improvement of this biotechnological approach.

Generation of Stable Influenza Virus Hemagglutinin through Structure-Guided Recombination.

Hemagglutinin (HA) is the major surface antigen of influenza virus and the most promising influenza vaccine immunogen. In 2013, the devastating H7N9 influenza virus was identified in China, which induced high mortality. The HA of this virus (H7) is relatively unstable, making it challenging to produce an effective vaccine. To improve the stability of HA protein from H7N9 influenza virus for better vaccine antigens without impairing immunogenicity, we recombined the HA from H7N9 (H7) with a more stable HA from H3N2 (H3) by structure-guided recombination, resulting in six chimeric HAs, FrA-FrF. Two of these chimeric HAs, FrB and FrC, exhibited proper hemagglutination activity and presented improved thermal stability compared to the original H7. Mice immunized with FrB and FrC elicited H7-specific antibodies comparable to those induced by parental H7, and the antisera collected from these immunized mice successfully inhibited H7N9 infection in a microneutralization assay. These results suggest that our structural-recombination approach can create stabilizing chimeric antigens while maintaining proper immunogenicity, which may not only benefit the construction of more stable HA vaccines to fight against H7N9 infection, but also facilitate effective vaccine improvements for other influenza viruses or infectious pathogens. In addition, this study also demonstrates the potential for better engineering of multimeric protein complexes like HA to achieve improved function, which are often immunologically or pharmaceutically important but difficult to modify.

Baculovirus IE2 Interacts with Viral DNA through Daxx To Generate an Organized Nuclear Body Structure for Gene Activation in Vero Cells.

Upon virus infection of a cell, the uncoated DNA is usually blocked by the host intrinsic immune system inside the nucleus. Although it is crucial for the virus to counteract the host intrinsic immune system and access its genome, little is known about how viruses can knock down host restriction and identify their blocked genomes for later viral gene activation and replication. We found that upon baculovirus transduction into Vero E6 cells, the invading viral DNA is trapped by the cellular death domain-associated protein (Daxx) and histone H3.3 in the nucleus, resulting in gene inactivation. IE2, a baculovirus transactivator, targets host Daxx through IE2 SUMO-interacting motifs (SIMs) to indirectly access viral DNA and forms unique nuclear body structures, which we term clathrate cage-like apparatus (CCLAs), at the early transduction stage. At the later transduction stage, CCLAs gradually enlarge, and IE2 continues to closely interact with viral DNA but no longer associates with Daxx. The association with Daxx is essential for IE2 CCLA formation, and the enlarged CCLAs are capable of transactivating viral but not chromosomal DNA of Vero E6 cells. Our study reveals that baculovirus IE2 counteracts the cellular intrinsic immune system by specifically targeting Daxx and H3.3 to associate with viral DNA indirectly and efficiently. IE2 then utilizes this association with viral DNA to establish a unique CCLA cellular nanomachinery, which is visible under light microscopy as an enclosed environment for proper viral gene expression.IMPORTANCE The major breakthrough of this work is that viral protein IE2 localizes and transactivates its own viral DNA through a most unlikely route, i.e., host proteins Daxx and H3.3, which are designed to efficiently restrict viral DNA from expression. By interacting with these host intrinsic immune factors, IE2 can thus target the viral DNA and then form a unique spherical nuclear body, which we name the CCLA, to enclose the viral DNA and necessary factors to assist in high-level transactivation. Our study represents one of the most complete investigations of nuclear body formation. In addition, so far only RNA or protein molecules have been reported as potential nucleators for initiating nuclear body formation; our study may represent the first example showing that DNA can be a nucleator for a new class of nuclear body formation.

Baculovirus as an efficient vector for gene delivery into mosquitoes.

Efficient gene delivery technologies play an essential role in the gene functional analyses that are necessary for basic and applied researches. Mosquitoes are ubiquitous insects, responsible for transmitting many deadly arboviruses causing millions of human deaths every year. The lack of efficient and flexible gene delivery strategies in mosquitoes are among the major hurdles for the study of mosquito biology and mosquito-pathogen interactions. We found that Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type baculovirus species, can efficiently transduce mosquito cells without viral propagation, allowing high level gene expression upon inducement by suitable promoters without obvious negative effects on cell propagation and viability. AcMNPV transduces into several mosquito cell types, efficiently than in commonly used mammalian cell lines and classical plasmid DNA transfection approaches. We demonstrated the application of this system by expressing influenza virus neuraminidase (NA) into mosquito hosts. Moreover, AcMNPV can transduce both larvae and adults of essentially all blood-sucking mosquito genera, resulting in bright fluorescence in insect bodies with little or no tissue barriers. Our experiments establish baculovirus as a convenient and powerful gene delivery vector in vitro and in vivo that will greatly benefit research into mosquito gene regulation, development and the study of mosquito-borne viruses.

Display of Porcine Epidemic Diarrhea Virus Spike Protein on Baculovirus to Improve Immunogenicity and Protective Efficacy.

A new variant of the porcine epidemic diarrhea virus (PEDV) is an emerging swine disease, killing considerable numbers of neonatal piglets in North America and Asia in recent years. To generate immunogens mimicking the complex spike (S) protein folding with proper posttranslational modification to mount a robust immune response against the highly virulent PEDV, two baculoviruses displaying the full-length S protein (S-Bac) and the S1 protein (S1-Bac) of the virulent Taiwan genotype 2b (G2b) PEDV Pintung 52 (PEDV-PT) strain were constructed. Intramuscular immunizations of mice and piglets with the S-Bac and S1-Bac demonstrated significantly higher levels of systemic anti-PEDV S-specific IgG, as compared with control group. Our results also showed that piglets in the S-Bac group elicited superior PEDV-specific neutralizing antibodies than those of the S1-Bac and control groups. The highly virulent PEDV-PT strain challenge experiment showed that piglets immunized with S-Bac and S1-Bac showed milder clinical symptoms with significantly less fecal viral shedding as compared with non-immunized control piglets. More importantly, piglets immunized with the S-Bac exhibited no to mild clinical signs, with a delayed, minimal viral shedding. Our results demonstrated that the S-Bac could serve as a safe, easy to manipulate, and effective vaccine candidate against the PEDV infection.

Graphene oxide sensitizes cancer cells to chemotherapeutics by inducing early autophagy events, promoting nuclear trafficking and necrosis.

Rationale: Cisplatin (CDDP) is a broad-spectrum anticancer drug but chemoresistance to CDDP impedes its wide use for cancer therapy. Autophagy is an event occurring in the cytoplasm and cytoplasmic LC3 puncta formation is a hallmark of autophagy. Graphene oxide (GO) is a nanomaterial that provokes autophagy in CT26 colon cancer cells and confers antitumor effects. Here we aimed to evaluate whether combined use of GO with CDDP (GO/CDDP) overcomes chemoresistance in different cancer cells and uncover the underlying mechanism. Methods: We treated different cancer cells with GO/CDDP and evaluated the cytotoxicity, death mechanism, autophagy induction and nuclear entry of CDDP. We further knocked down genes essential for autophagic flux and deciphered which step is critical to nuclear import and cell death. Finally, we performed immunoprecipitation, mass spectrometry and immunofluorescence labeling to evaluate the association of LC3 and CDDP. Results: We uncovered that combination of GO and CDDP (GO/CDDP) promoted the killing of not only CT26 cells, but also ovarian, cervical and prostate cancer cells. In the highly chemosensitized Skov-3 cells, GO/CDDP significantly enhanced concurrent nuclear import of CDDP and autophagy marker LC3 and elevated cell necrosis, which required autophagy initiation and progression but did not necessitate late autophagy events (e.g., autophagosome completion and autolysosome formation). The GO/CDDP-elicited nuclear trafficking and cell death also required importin α/ß, and LC3 also co-migrated with CDDP and histone H1/H4 into the nucleus. In particular, GO/CDDP triggered histone H4 acetylation in the nucleus, which could decondense the chromosome and enable CDDP to more effectively access chromosomal DNA to trigger cell death. Conclusion: These findings shed light on the mechanisms of GO/CDDP-induced chemosensitization and implicate the potential applications of GO/CDDP to treat multiple cancers.

Global Screening of Antiviral Genes that Suppress Baculovirus Transgene Expression in Mammalian Cells.

Although baculovirus has been used as a safe and convenient gene delivery vector in mammalian cells, baculovirus-mediated transgene expression is less effective in various mammalian cell lines. Identification of the negative regulators in host cells is necessary to improve baculovirus-based expression systems. Here, we performed high-throughput shRNA library screening, targeting 176 antiviral innate immune genes, and identified 43 host restriction factor genes in a human A549 lung carcinoma cell line. Among them, suppression of receptor interaction protein kinase 1 (RIP1, also known as RIPK1) significantly increased baculoviral transgene expression without resulting in significant cell death. Silencing of RIP1 did not affect viral entry or cell viability, but it did inhibit nuclear translocation of the IRF3 and NF-κB transcription factors. Also, activation of downstream signaling mediators (such as TBK1 and IRF7) was affected, and subsequent interferon and cytokine gene expression levels were abolished. Further, Necrostatin-1 (Nec-1)-an inhibitor of RIP1 kinase activity-dramatically increased baculoviral transgene expression in RIP1-silenced cells. Using baculovirus as a model system, this study presents an initial investigation of large numbers of human cell antiviral innate immune response factors against a "nonadaptive virus." In addition, our study has made baculovirus a more efficient gene transfer vector for some of the most frequently used mammalian cell systems.

Exploring the Mechanism Responsible for Cellulase Thermostability by Structure-Guided Recombination.

Cellulases from Bacillus and Geobacillus bacteria are potentially useful in the biofuel and animal feed industries. One of the unique characteristics of these enzymes is that they are usually quite thermostable. We previously identified a cellulase, GsCelA, from thermophilic Geobacillus sp. 70PC53, which is much more thermostable than its Bacillus homolog, BsCel5A. Thus, these two cellulases provide a pair of structures ideal for investigating the mechanism regarding how these cellulases can retain activity at high temperature. In the present study, we applied the SCHEMA non-contiguous recombination algorithm as a novel tool, which assigns protein sequences into blocks for domain swapping in a way that lessens structural disruption, to generate a set of chimeric proteins derived from the recombination of GsCelA and BsCel5A. Analyzing the activity and thermostability of this designed library set, which requires only a limited number of chimeras by SCHEMA calculations, revealed that one of the blocks may contribute to the higher thermostability of GsCelA. When tested against swollen Avicel, the highly thermostable chimeric cellulase C10 containing this block showed significantly higher activity (22%-43%) and higher thermostability compared to the parental enzymes. With further structural determinations and mutagenesis analyses, a 310 helix was identified as being responsible for the improved thermostability of this block. Furthermore, in the presence of ionic calcium and crown ether (CR), the chimeric C10 was found to retain 40% residual activity even after heat treatment at 90°C. Combining crystal structure determinations and structure-guided SCHEMA recombination, we have determined the mechanism responsible for the high thermostability of GsCelA, and generated a novel recombinant enzyme with significantly higher activity.

Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning.

Baculoviruses have gained popularity as pest control agents and for protein production in insect systems. These viruses are also becoming popular for gene expression, tissue engineering and gene therapy in mammalian systems. Baculovirus infection triggers a heat shock response, and this response is crucial for its successful infection of host insect cells. However, the viral protein(s) or factor(s) that trigger this response are not yet clear. Previously, we revealed that IE2-an early gene product of the baculovirus-could form unique nuclear bodies for the strong trans-activation of various promoters in mammalian cells. Here, we purified IE2 nuclear bodies from Vero E6 cells and investigated the associated proteins by using mass spectrometry. Heat shock proteins (HSPs) were found to be one of the major IE2-associated proteins. Our experiments show that HSPs are greatly induced by IE2 and are crucial for the trans-activation function of IE2. Interestingly, blocking both heat shock protein expression and the proteasome pathway preserved the IE2 protein and its nuclear body structure, and revived its function. These observations reveal that HSPs do not function directly to assist the formation of the nuclear body structure, but may rather protect IE2 from proteasome degradation. Aside from functional studies in mammalian cells, we also show that HSPs were stimulated and required to determine IE2 protein levels, in insect cells infected with baculovirus. Upon inhibiting the expression of heat shock proteins, baculovirus IE2 was substantially suppressed, resulting in a significantly suppressed viral titer. Thus, we demonstrate a unique feature in that IE2 can function in both insect and non-host mammalian cells to stimulate HSPs, which may be associated with IE2 stabilization and lead to the protection of the its strong gene activation function in mammalian cells. On the other hand, during viral infection in insect cells, IE2 could also strongly stimulate HSPs and ultimately affect viral replication.