At least three major antigenic dengue 2 virus proteins were recognized by pooled dengue fever patients' sera in infected Aedes albopictus (C6/36) mosquito cells. Dengue virus envelope (E), premembrane (PrM) and non-structural protein 1 (NS 1) dimer were detected beginning on day 3 postinfection in both the cell membrane and cytosolic fractions. Using the patients' sera, the presence of antigenic intermediate core protein (C)-PrM and NS1-non-structural protein 2a (NS2a) in the cytoplasmic fraction of dengue 2 virus infected cells was revealed. The presence of a approximately 92 and approximately 84 kDa NS 1 dimer in the membrane (NS 1m) and cytosolic (NS 1c) fractions of C6/36 cells, respectively, was also recognized. Using individual patient's serum, it was further confirmed that all patients' sera contained antibodies that specifically recognized E, NS 1 and PrM present in the dengue 2 virus-infected cell membrane fractions, suggesting that these glycosylated virus proteins were the main antigenic proteins recognized in vivo. Detection of dengue 2 virus C antibody in some patients further suggested that C could be antigenic if presented in vivo.
The nucleocapsid (NP) protein of Newcastle disease virus (NDV) self-assembled in Escherichia coli as ring-like and herringbone-like particles. Several chimeric NP proteins were constructed in which the antigenic regions of the hemagglutinin-neuraminidase (HN) and fusion (F) proteins of NDV, myc epitope, and six histidines (a hexa-His tag) were linked to the C-terminus of the NP monomer. These chimeric proteins were expressed efficiently in soluble form in E. coli as detected by Western blot analysis. Electron microscopy of the purified products revealed that they self-assembled into ring-like particles. These chimeric particles exhibited antigenicity of the myc epitope, suggesting that the foreign sequences were exposed on the surface of the particles. Chickens inoculated with the chimeric particles mounted an immune response against NDV, suggesting the possibility of use of the ring-like particle as a carrier of immunogens in subunit vaccines and immunological reagents.
The nucleocapsid (NP) and phospho-(P) proteins of paramyxoviruses are involved in transcription and replication of the viral genome. An in vitro protein binding assay was used to investigate the regions on NP protein that interact with the P protein of Newcastle disease virus (NDV). Truncated NP mutants were first immobilised on a solid phase and then interacted with radio-labelled [(35)S]-P protein synthesised in rabbit reticulocyte. The interaction affinity was quantitated by measuring the radioactivity that was retained on the solid phase. Using this approach, a highly interactive region was identified to be resided at the first 25 amino acids of NP N-terminus. The interaction between these two proteins remained strong even with the removal of 114 amino acids from the C-terminal end of NP. However, it is possible that the 49 amino acids at the C-terminal end might have another contact region for P protein, which is not as critical as the N-terminal end. The interaction regions mapped in this study are significantly different from the other two paramyxoviruses: Sendai and measles viruses in which the C-termini of their NP proteins play an important role in binding to the P.
A single chain variable fragment (scFv) specific towards B. pseudomallei exotoxin had previously been generated from an existing hybridoma cell line (6E6AF83B) and cloned into the phage display vector pComb3H. In this study, the scFv was subcloned into the pComb3X vector to facilitate the detection and purification of expressed antibodies. Detection was facilitated by the presence of a hemagglutinin (HA) tag, and purification was facilitated by the presence of a histidine tag. The culture was grown at 30 degrees C until log phase was achieved and then induced with 1 mM IPTG in the absence of any additional carbon source. Induction was continued at 30 degrees C for five h. The scFv was discerned by dual processes-direct enzyme-linked immunosorbent assays (ELISA), and Western blotting. When compared to E. coli strains ER2537 and HB2151, scFv expression was observed to be highest in the E. coli strain Top10F'. The expressed scFv protein was purified via nickel-mediated affinity chromatography and results indicated that two proteins a 52 kDa protein, and a 30 kDa protein were co-purified. These antibodies, when blotted against immobilized exotoxin, exhibited significant specificity towards the exotoxin, compared to other B. pseudomallei antigens. Thus, these antibodies should serve as suitable reagents for future affinity purification of the exotoxin.
The yeast two-hybrid system has been used to identify domains of the Newcastle disease virus (NDV) phosphoprotein (P) involved in self-association and interaction with the nucleocapsid protein (NP). Deletion analysis was used to map the domain(s) of the P protein involved in P:P and P:NP interactions. The C-terminal 45 amino acids (residues 247-291) were shown to play a major role in both of the interactions. Comparison of these findings with other reports suggests that paramyxoviruses are different with respect to interaction domain(s) between these two essential viral proteins involved in genome replication.
In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with Ni2+ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was 1.26% and 5.56%, respectively. It was demonstrated that EBA achieved the highest final protein yield of 9.6% with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.
Directed evolution of nucleotide libraries using recombination or mutagenesis is an important technique for customizing catalytic or biophysical traits of proteins. Conventional directed evolution methods, however, suffer from cumbersome digestion and ligation steps. Here, we describe a simple method to increase nucleotide diversity using single-stranded DNA (ssDNA) as a starting template. An initial PCR amplification using phosphorylated primers with overlapping regions followed by treatment with lambda exonuclease generates ssDNA templates that can then be annealed via the overlap regions. Double-stranded DNA (dsDNA) is then generated through extension with Klenow fragment. To demonstrate the applicability of this methodology for directed evolution of nucleotide libraries, we generated both gene shuffled and regional mutagenesis synthetic antibody libraries with titers of 2×108 and 6×107, respectively. We conclude that our method is an efficient and convenient approach to generate diversity in nucleic acid based libraries, especially recombinant antibody libraries.
Global resurgence of dengue virus infections in many of the tropical and subtropical countries is a major concern. Therefore, there is an urgent need for the development of successful drugs that are both economical and offer a long-lasting protection. The viral NS2B-NS3 serine protease (NS2B-NS3pro) is a promising target for the development of drug-like inhibitors, which are not available at the moment. In this study, we report retrocyclin-1 (RC-1) production in E. coli as a recombinant peptide to test against dengue NS2B-NS3pro.
Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus that has caused multiple unprecedented and re-emerging outbreaks in both tropical and temperate countries. Despite ongoing research efforts, the underlying factors involved in facilitating CHIKV replication during early infection remains ill-characterized. The present study serves to identify host proteins modulated in response to early CHIKV infection using a proteomics approach.
Direct protein delivery into the cytosol of mammalian cells by invasive Escherichia coli (E. coli) bacterial vector will bypass the need to achieve nuclear entry and transcription of DNA, a major hurdle that is known to seriously limit gene transfer. The bacterial vector is induced to express the protein during its growth phase, before presentation for entry into mammalian cells and release of its content into the cellular environment. For this class of vector, crossing the plasma membrane becomes the primary step that determines the success of protein delivery. Yet, how the mechanics of protein expression within the vector affect its entry into the host is poorly understood. We found the vector's effectiveness to enter HeLa cells diminished together with its viability when phage N15 protelomerase (TelN) expression was induced continuously in the invasive E. coli despite producing an abundant amount of functional protein. By comparison, shorter induction, even as little as 3 hr, produced sufficient amounts of functional TelN and showed more effective invasion of HeLa cells, comparable to that of uninduced invasive E. coli. These results demonstrate that brief induction of protein expression during vector growth is essential for optimal entry into mammalian cells, an important step for achieving bacteria-mediated protein delivery.
Nodamura virus (NoV) B2, a suppressor of RNA interference, binds double stranded RNAs (dsRNAs) and small interfering RNAs (siRNAs) corresponding to Dicer substrates and products. Here, we report that the amino terminal domain of NoV B2 (NoV B2 79) specifically binds siRNAs but not dsRNAs. NoV B2 79 oligomerizes on binding to 27 nucleotide siRNA. Mutation of the residues phenylalanine49 and alanine60 to cysteine and methionine, respectively enhances the RNA binding affinity of NoV B2 79. Circular dichroism spectra demonstrated that the wild type and mutant NoV B2 79 have similar secondary structure conformations.
INTRODUCTION: Dengue virus (DENV) is principally transmitted by the Aedes aegypti mosquito. To date, mosquito population control remains the key strategy for reducing the continuing spread of DENV. The focus on the development of new vector control strategies through an understanding of the mosquito-virus relationship is essential, especially targeting the midgut, which is the first mosquito organ exposed to DENV infection.
METHODOLOGY: A cDNA library derived from female adult A. aegypti mosquito midgut cells was established using the switching mechanism at the 5' end of the RNA transcript (SMART), in combination with a highly potent recombination machinery of Saccharomyces cerevisiae. Gal4-based yeast two-hybrid (Y2H) assays were performed against DENV-2 proteins (E, prM, M, and NS1). Mammalian two-hybrid (M2H) and double immunofluorescence assays (IFA) were conducted to validate the authenticity of the three selected interactions.
RESULTS: The cDNA library was of good quality based on its transformation efficiency, cell density, titer, and the percentage of insert size. A total of 36 midgut proteins interacting with DENV-2 proteins were identified, some involved in nucleic acid transcription, oxidoreductase activity, peptidase activity, and ion binding. Positive outcomes were obtained from the three selected interactions validated using M2H and double IFA assays.
CONCLUSIONS: The identified proteins have different biological activities that may aid in the virus replication pathway. Therefore, the midgut cDNA library is a valuable tool for identifying DENV-2 interacting proteins. The positive outcomes of the three selected proteins validated supported the quality of the cDNA library and the robustness of the Y2H mechanisms.
Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.
Newcastle disease virus remains a constant threat in commercial poultry farms despite intensive vaccination programs. Outbreaks attributed to ND can escalate and spread across farms and states contributing to major economic loss in poultry farms.
Our understanding of the proteolytic processing events at the NS1-2A junction in the flavivirus polyprotein has not markedly progressed since the early work conducted on dengue virus (DENV). This work identified an octapeptide sequence located immediately upstream of the cleavage site thought to be important in substrate recognition by an as yet unknown, endoplasmic reticulum-resident host protease. Of the eight amino acid recognition sequence, the highly conserved residues at positions P1, P3, P5, P7 and P8 (with respect to N-terminus of NS2A) are particularly sensitive to amino acid substitutions in terms of DENV NS1-NS2A cleavage efficiency; however, the role of the octapeptide in efficient NS1 and NS2A production of other flaviviruses has not been experimentally addressed.
The human alphacoronaviruses HCoV-NL63 and HCoV-229E are commonly associated with upper respiratory tract infections (URTI). Information on their molecular epidemiology and evolutionary dynamics in the tropical region of southeast Asia however is limited. Here, we analyzed the phylogenetic, temporal distribution, population history, and clinical manifestations among patients infected with HCoV-NL63 and HCoV-229E. Nasopharyngeal swabs were collected from 2,060 consenting adults presented with acute URTI symptoms in Kuala Lumpur, Malaysia, between 2012 and 2013. The presence of HCoV-NL63 and HCoV-229E was detected using multiplex polymerase chain reaction (PCR). The spike glycoprotein, nucleocapsid, and 1a genes were sequenced for phylogenetic reconstruction and Bayesian coalescent inference. A total of 68/2,060 (3.3%) subjects were positive for human alphacoronavirus; HCoV-NL63 and HCoV-229E were detected in 45 (2.2%) and 23 (1.1%) patients, respectively. A peak in the number of HCoV-NL63 infections was recorded between June and October 2012. Phylogenetic inference revealed that 62.8% of HCoV-NL63 infections belonged to genotype B, 37.2% was genotype C, while all HCoV-229E sequences were clustered within group 4. Molecular dating analysis indicated that the origin of HCoV-NL63 was dated to 1921, before it diverged into genotype A (1975), genotype B (1996), and genotype C (2003). The root of the HCoV-229E tree was dated to 1955, before it diverged into groups 1-4 between the 1970s and 1990s. The study described the seasonality, molecular diversity, and evolutionary dynamics of human alphacoronavirus infections in a tropical region.
Positive transcription elongation factor b (P-TEFb), which comprises cyclin-dependent kinase 9 (CDK9) kinase and cyclin T subunits, is an essential kinase complex in human cells. Phosphorylation of the negative elongation factors by P-TEFb is required for productive elongation of transcription of protein-coding genes by RNA polymerase II (pol II). In addition, P-TEFb-mediated phosphorylation of the carboxyl-terminal domain (CTD) of the largest subunit of pol II mediates the recruitment of transcription and RNA processing factors during the transcription cycle. CDK9 also phosphorylates p53, a tumor suppressor that plays a central role in cellular responses to a range of stress factors. Many viral factors affect transcription by recruiting or modulating the activity of CDK9. In this review, we will focus on how the function of CDK9 is regulated by viral gene products. The central role of CDK9 in viral life cycles suggests that drugs targeting the interaction between viral products and P-TEFb could be effective anti-viral agents.
Rotavirus B (RVB) infection in cattle is poorly understood. The objective of this study was to describe the epidemiological features of repeated outbreaks of epidemic diarrhea due to RVB infection in adult cattle on a large dairy farm complex in Japan. In October 2002, approximately 550 adult cows and approximately 450 in February 2005 had acute watery diarrhea at several farms on the complex. Four months before the first outbreak, RVB antibody-positive rates at subsequently affected farms were significantly lower than at non-affected farms (30% to 32% versus 61% to 67%). During the acute phase of both outbreaks, RVB antibody-positive rates in diarrheal cows tested were as low as 15% to 26%. Most of the farms affected in the second outbreak were also involved in the first outbreak. Some adult cows with RVB diarrhea in the first outbreak showed not only RVB seroresponse, but also RVB shedding in the second outbreak, although none of these cows developed diarrhea. Nucleotide sequences of the VP7 and VP4 genes revealed a close relationship between RVB strains in both outbreaks. Taken together, these results indicate that outbreaks of epidemic RVB diarrhea in adult cows might be influenced by herd immunity and could occur repeatedly at the same farms over several years. To our knowledge, this is the first report on repeated RVB infections in the same cattle.
Enterovirus A71 (EV-A71) is a neurotropic enterovirus that uses heparan sulfate as an attachment receptor. The molecular determinants of EV-A71-heparan sulfate interaction are unknown. With In silico heparin docking and mutagenesis of all possible lysine residues in VP1, we identified that K162, K242 and K244 are responsible for heparin interaction and inhibition. EV-A71 mutants with K242A and K244A rapidly acquired compensatory mutations, T100K or E98A, and Q145R-T237N respectively, which restored the heparin-binding phenotype. Both VP1-98 and VP1-145 modulates heparin binding. Heparin-binding phenotype was completely abolished with VP1-E98-E145, but was restored by an E98K or E145Q substitution. During cell culture adaptation, EV-A71 rapidly acquired K98 or Q/G145 to restore the heparin-binding phenotype. Together with next-generation sequencing analysis, our results implied that EV-A71 has high genetic plasticity by modulating positively-charged residues at the five-fold axis during in vitro heparin adaptation. Our finding has impact on EV-A71 vaccine production, evolutionary studies and pathogenesis.
The discovery of highly active antiretroviral therapy (HAART) in 1996 has significantly reduced the global mortality and morbidity caused by the acquired immunodeficiency syndrome (AIDS). However, the therapeutic strategy of HAART that targets multiple viral proteins may render off-target toxicity and more importantly results in drug-resistant escape mutants. These have been the main challenges for HAART and refinement of this therapeutic strategy is urgently needed. Antibody-mediated treatments are emerging therapeutic modalities for various diseases. Most therapeutic antibodies have been approved by Food and Drug Administration (FDA) mainly for targeting cancers. Previous studies have also demonstrated the promising effect of therapeutic antibodies against HIV-1, but there are several limitations in this therapy, particularly when the viral targets are intracellular proteins. The conventional antibodies do not cross the cell membrane, hence, the pathogenic intracellular proteins cannot be targeted with this classical therapeutic approach. Over the years, the advancement of antibody engineering has permitted the therapeutic antibodies to comprehensively target both extra- and intra-cellular proteins in various infections and diseases. This review aims to update on the current progress in the development of antibody-based treatment against intracellular targets in HIV-1 infection. We also attempt to highlight the challenges and limitations in the development of antibody-based therapeutic modalities against HIV-1.