[Show abstract][Hide abstract] ABSTRACT: Background
Fitness costs and slower disease progression are associated with a cytolytic T lymphocyte (CTL) escape mutation T242N in Gag in HIV-1-infected individuals carrying HLA-B*57/5801 alleles. However, the impact of different context in diverse HIV-1 strains on the fitness costs due to the T242N mutation has not been well characterized. To better understand the extent of fitness costs of the T242N mutation and the repair of fitness loss through compensatory amino acids, we investigated its fitness impact in different transmitted/founder (T/F) viruses.ResultsThe T242N mutation resulted in various levels of fitness loss in four different T/F viruses. However, the fitness costs were significantly compromised by preexisting compensatory amino acids in (Isoleucine at position 247) or outside (glutamine at position 219) the CTL epitope. Moreover, the transmitted T242N escape mutant in subject CH131 was as fit as the revertant N242T mutant and the elimination of the compensatory amino acid I247 in the T/F viral genome resulted in significant fitness cost, suggesting the fitness loss caused by the T242N mutation had been fully repaired in the donor at transmission. Analysis of the global circulating HIV-1 sequences in the Los Alamos HIV Sequence Database showed a high prevalence of compensatory amino acids for the T242N mutation and other T cell escape mutations.Conclusions
Our results show that the preexisting compensatory amino acids in the majority of circulating HIV-1 strains could significantly compromise the fitness loss due to CTL escape mutations and thus increase challenges for T cell based vaccines.
[Show abstract][Hide abstract] ABSTRACT: Traditionally, infectivity of a trivalent live attenuated influenza vaccines (LAIVs) is titrated by determining the 50% egg infectious dose assay (EID50) or plaque forming units (PFU), which requires specific monoclonal antibodies to neutralize two strains while estimating the titer of the non-neutralized strain. Compared to this time-consuming, laborious, subjective and variable process, reverse transcription-quantitative real-time PCR (RT-qPCR) technology has advantages of rapidity, sensitivity, reproducibility and reduced contamination, thus has been applied widely for detecting pathogens and measuring viral titers. In this study, the critical harvest time was determined to be 18 h post-infection (hpi) for type A influenza and 12 hpi for type B influenza, but no significant difference between titers at 12 hpi and 18 hpi for the type B strain was observed. In conclusion, trivalent LAIVs can be titrated simultaneously within 24 h by this one-step RT-qPCR assay, which yielded titers comparable to those obtained by the traditional EID50 assay. Therefore, the RT-qPCR assay may be used as a highly specific, sensitive, precise and rapid alternative to the EID50 assay for titering LAIVs.
Human Vaccines and Therapeutics 11/2014; 10(12):e34453. · 3.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BST-2 blocks the particle release of various enveloped viruses including HIV-1, and this antiviral activity is dependent on the topological arrangement of its four structural domains. Several functions of the cytoplasmic tail (CT) of BST-2 have been previously discussed, but the exact role of this domain remains to be clearly defined. In this study, we investigated the impact of truncation and commonly-used tags addition into the CT region of human BST-2 on its intracellular trafficking and signaling as well as its anti-HIV-1 function. The CT-truncated BST-2 exhibited potent inhibition on Vpu-defective HIV-1 and even wild-type HIV-1. However, the N-terminal HA-tagged CT-truncated BST-2 retained little antiviral activity and dramatically differed from its original protein in the cell surface level and intracellular localization. Further, we showed that the replacement of the CT domain with a hydrophobic tag altered BST-2 function possibly by preventing its normal vesicular trafficking. Notably, we demonstrated that a positive charged motif "KRXK" in the conjunctive region between the cytotail and the transmembrane domain which is conserved in primate BST-2 is important for the protein trafficking and the antiviral function. These results suggest that although the CT of BST-2 is not essential for its antiviral activity, the composition of residues in this region may play important roles in its normal trafficking which subsequently affected its function. These observations provide additional implications for the structure-function model of BST-2.
PLoS ONE 10/2014; 9(10):e111422. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
All lentiviruses except equine infectious anemia virus (EIVA) antagonize antiviral family APOBEC3 (A3) proteins of the host through viral Vif proteins. The mechanism by which Vif of human, simian or feline immunodeficiency viruses (HIV/SIV/FIV) suppresses the corresponding host A3s has been studied extensively.ResultsHere, we determined that bovine immunodeficiency virus (BIV) and maedi-visna virus (MVV) Vif proteins utilize the Cullin (Cul)-ElonginB (EloB)-ElonginC (EloC) complex (BIV Vif recruits Cul2, while MVV Vif recruits Cul5) to degrade Bos taurus (bt)A3Z2-Z3 and Ovis aries (oa)A3Z2-Z3, respectively, via a proteasome-dependent but a CBF-ß-independent pathway. Mutation of the BC box in BIV and MVV Vif, C-terminal hydrophilic replacement of btEloC and oaEloC and dominant-negative mutants of btCul2 and oaCul5 could disrupt the activity of BIV and MVV Vif, respectively. While the membrane-permeable zinc chelator TPEN could block BIV Vif-mediated degradation of btA3Z2-Z3, it had minimal effects on oaA3Z2-Z3 degradation induced by MVV Vif, indicating that Zn is important for the activity of BIV Vif but not MVV Vif. Furthermore, we identified a previously unreported zinc binding loop [C-x1-C-x1-H-x19-C] in the BIV Vif upstream BC box which is critical for its degradation activity.ConclusionsA novel zinc binding loop was identified in the BIV Vif protein that is important for the E3 ubiquination activity, suggesting that the degradation of btA3Z2-Z3 by BIV and that of oaA3Z2-Z3 by MVV Vif may need host factors other than CBF-ß.
[Show abstract][Hide abstract] ABSTRACT: Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) have caused severe epidemics of hand, foot and mouth disease (HFMD) in the Asia Pacific in recent years, particularly in infants and young children. This disease has become a serious public health problem, as no vaccines or antiviral drugs have been approved for EV71 and CA16 infections. In this study, we compared four monovalent vaccines, including formalin-inactivated EV71 virus (iEV71), EV71 virus-like particles (VLPs) (vEV71), formalin-inactivated CVA16 virus (iCVA16) and CVA16 VLPs (vCVA16), along with two bivalent vaccines, including equivalent doses of formalin-inactivated EV71+CVA16 virus (iEV71+iCVA16) and EV71+CVA16 VLPs (vEV71+vCVA16). The IgG titers and neutralization antibodies titers demonstrated that there are no immune interference exists between the two immunogens of EV71 and CVA16. IgG subclass isotyping revealed that IgG1 and IgG2b were induced primarily in all vaccine groups. Furthermore, cross-neutralization antibodies were elicited in mouse sera against other sub-genotypes of EV71 and CVA16. In vivo challenge experiments showed that the immune sera from vaccinated animals could confer passive protection to newborn mice against lethal challenge with 14 LD50 of EV71 and 50 LD50 of CVA16. Our results indicated that bivalent vaccination is promising for HFMD vaccine development. With the advantage of having a better safety profile than inactivated virus vaccines, VLPs should be used to combine both EV71 and CVA16 antigens as a candidate vaccine for prevention of HFMD virus transmission.
[Show abstract][Hide abstract] ABSTRACT: Streptococcus pneumoniae is a major cause of infectious disease and complications worldwide, such as pneumonia, otitis media, bacteremia and meningitis. New generation protein-based pneumococcal vaccines are recognized as alternative vaccine candidates. Pneumolysin (Ply) is a cholesterol-dependent cytolysin produced by all clinical isolates of S. pneumoniae. Our research group previously developed a highly detoxified Ply mutant designated Plym2 by replacement of two animo acids (C428G and W433F). Exhibiting undetectable levels of cytotoxicity, Plym2 could still elicit high titer neutralizing antibodies against the native toxin. However, evaluation of the active immunoprotective effects of Plym2 by subcutaneous immunization and lethal challenge with S. pneumoniae in mice did not yield favorable results. In the present work, we confirmed the previous observations by using passive immunization and systemic challenge. Results of the passive immunization were consistent with those of active immunization. Further experiments were conducted to explain the inability of high titer neutralizing antibodies against Ply to protect mice from S. pneumoniae challenge. Pneumococcal Ply is known to be the major factor responsible for the induction of inflammation that benefits the host. Proinflammatory cytokines facilitate the clearance of invaders by the recruitment and activation of leukocytes at the early infection stage. We demonstrated that Plym2 could induce proinflammatory cytokines similarly to wild-type Ply. A systemic infection model was used to clarify that Plym2 lacking cytolytic activity could protect mice from intraperitoneal challenge directly, while antibodies to the mutant had no effect. Therefore, the protective function of Plym2 may be due to its induction of proinflammatory cytokines. When used in the systemic infection model, Plym2 antibodies may block the induction of proinflammatory cytokines by Ply. These findings demonstrate that a Ply-based vaccine would not be an effective primary vaccine component, but it may be beneficial as an adjuvant to stimulate cytokine production.
[Show abstract][Hide abstract] ABSTRACT: Hand, foot and mouth disease (HFMD) is a common pediatric illness mainly caused by infection with enterovirus 71 (EV71) and coxsackievirus A16 (CA16). The frequent HFMD outbreaks have become a serious public health problem. Currently, no vaccine or antiviral drug for EV71/CA16 infections has been approved. In this study, a two-step screening platform consisting of reporter virus-based assays and cell viability‑based assays was developed to identify potential inhibitors of EV71/CA16 infection. Two types of reporter viruses, a pseudovirus containing luciferase-encoding RNA replicons encapsidated by viral capsid proteins and a full-length reporter virus containing enhanced green fluorescent protein, were used for primary screening of 400 highly purified natural compounds. Thereafter, a cell viability-based secondary screen was performed for the identified hits to confirm their antiviral activities. Three compounds (luteolin, galangin, and quercetin) were identified, among which luteolin exhibited the most potent inhibition of viral infection. In the cell viability assay and plaque reduction assay, luteolin showed similar 50% effective concentration (EC50) values of about 10 μM. Luteolin targeted the post-attachment stage of EV71 and CA16 infection by inhibiting viral RNA replication. This study suggests that luteolin may serve as a lead compound to develop potent anti-EV71 and CA16 drugs.
[Show abstract][Hide abstract] ABSTRACT: Hand, foot and mouth disease, associated with enterovirus 71 (EV71) infections, has recently become an important public health issue throughout the world. Serum neutralizing antibodies are major indicators of EV71 infection and protective immunity. However, the potential for cross-reactivity of neutralizing antibodies for different EV71 genotypes and subgenotypes is unclear. Here we measured the cross-reactive neutralizing antibody titers against EV71 of different genotypes or subgenotypes in sera collected from EV71-infected children and vaccine-inoculated children in a phase III clinical trial (ClinicalTrials.gov Identifier: NCT01636245) using a new pseudovirus-based neutralization assay. Antibodies induced by EV71-C4a were cross-reactive for different EV71 genotypes, demonstrating that C4a is a good candidate strain for an EV71 vaccine. Our study also demonstrated that this new assay is practical for analyses of clinical samples from epidemiological and vaccine studies.
PLoS ONE 06/2014; 9(6):e100545. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Eliciting efficient broadly neutralizing antibodies (BnAbs) is an important goal that has yet to be achieved for human immunodeficiency type 1 (HIV-1) vaccine development, although they are rarely produced in virus-infected individuals. In particular, inducing specific neutralizing antibodies to the gp41 membrane proximal external region (MPER) has proven a difficult task. In this study, we introduce Norovirus P particles as a new platform to display the MPER epitope of HIV-1 as a vaccine with the aim of enhancing immune responses. The results showed that HIV-1 chimeric P particles were capable of inducing MPER-specific antibody responses in immunized guinea pigs, although only weakly neutralizing activity could be detected. These findings are consistent with other previous studies which have also focused on the well-studied 2F5 and 4E10 BnAbs. Our findings provide an alternate strategy for design of vaccines against HIV-1. However, great challenges remain in the effort to develop vaccines that can induce efficient HIV-1 neutralizing antibodies.
Protein and Peptide Letters 06/2014; · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rabies virus (RV) causes a fatal infectious disease requiring efficient post-exposure prophylaxis (PEP), which includes a rabies vaccine and rabies immunoglobulin (RIG). The single-chain antibody variable fragment (scFv), a small engineered antibody fragment derived from an antibody variable heavy chain and light chain, has the potential to replace the current application of RIG. In previous studies, we constructed and evaluated an anti-rabies virus G protein scFv (FV57) based on the monoclonal antibody CR57. Of the five cysteines in FV57, four are linked in intra-chain disulfide bonds (Cys-VH28/Cys-VH98 and Cys-VL16/Cys-VL84), and one is free (Cys-VL85). However, the thiol in Cys-VL85 neighboring Cys-VL84 in the CDR3 of the light chain is likely to mismatch with the thiol in Cys-VL16 during the renaturing process. In order to study effects of the mismatched disulfide bond, Cys-VL85 and Cys-VL84 of FV57 were mutated to serine to construct mutants FV57VL85S and FV57VL84S. Furthermore, the disulfide bonds in the light chain of FV57, FV57VL85S and FV57VL84S were deleted by mutating Cys-VL16 to serine. All mutants were prepared and evaluated along with the original FV57. The results indicated that the mismatched disulfide bond of FV57 linking the light chain FR1 and CDR3 would confer deleterious negative effects on its activity against RV, likely due to spatial hindrance in the light chain CDR3. Moreover, avoidance of the disulfide bond mismatch provided an additional 30% protective efficacy against RV infection in the mouse RV challenge model. Thus, modifications of FV57 to eliminate the disulfide bond mismatch may provide a candidate therapeutic agent for effective PEP against rabies.
[Show abstract][Hide abstract] ABSTRACT: Adenoviruses (Ads) are nonenveloped DNA viruses that have been extensively studied and used as vectors for gene therapy and several potential vaccines. There are 57 Ad serotypes in seven species (A-G), and Ad neutralizing antibodies (NAbs) titers can vary by serotypes and geographic location. Until now serotype- and species-specific antibodies have been detected by neutralization or hemagglutination inhibition assays. These expensive and cumbersome methods of adenovirus typing have mainly been used in epidemiological studies. Our prior work demonstrated that NAbs against the fiber protein are commonly generated during natural Ad infection in humans and the trimeric knob is preferentially recognized by fiber-induced NAbs. In this study, we expressed nine trimeric knob proteins from representative Ad serotypes of human Ad (HAdV)-A to -F in Escherichia coli and found no cross-reactivity of these recombinant proteins with rabbit hyperimmune sera (among HAdV-A to -F or within HAdV-C). Results of the ELISA based on Ad2 and Ad5 (both HAdV-C) knob proteins were consistent with those of neutralization assays, indicating that the trimeric knob protein would be a good candidate antigen for detecting Ad serotype-specific NAbs in sera from naturally infected subjects. We also demonstrated the primary seroepidemiology of nine Ad serotypes in 274 children samples by the knob-based ELISA. These results will have potential implications for epidemiology of Ad serotypes and future development of Ad-based vaccines and gene therapy.
Journal of General Virology 04/2014; · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Breast cancers especially in late and metastatic stages remain refractory to treatment despite advances in surgical techniques and chemotherapy. Suicide gene therapy based on adenoviral technology will be promising strategies for such advanced diseases. We previously showed that co-expression of herpes simplex virus thymidine kinase (HSV-TK) and Escherichia coli nitroreductase (Coli.NTR) by an hTERT-driven adenovirus vector resulted in additive anti-tumor effects in breast cancer cells in vitro and in vivo. As many tumor tissue and cancer cells express low level of coxsackie-adenovirus receptor (CAR), which is the functional receptor for the fiber protein of human adenovirus serotype 5 (Ad5), novel Ad5 vectors containing genetically modifi ed fiber are attractive vehicles for achieving targeted gene transfer and improving suicide gene expression in these cancer cells. In the present study, we first built a simplified Ad5 vector platform for fiber modification and quick detection for gene transfer. Then a fiber-modified adenovirus vector containing an RGD motif in the HI loop of the fiber knob was constructed. After recombined with HSV-TK and Coli.NTR gene, this fiber-modified Ad5 vector (Ad-RGD-hT-TK/NTR) was compared with that of our previously constructed Ad5 vector (Ad-hT-TK/NTR) for its therapeutic effects in human breast cancer cell lines. The anti-tumor activity of Ad-RGD-hT-TK/NTR was significantly enhanced compared with Ad-hT-TK/NTR both in vitro and in vivo. This new vector platform provided a robust and simplified approach for capsid modification, and the fiber-modified Ad5 with double suicide genes under the control of hTERT promoter would be a useful gene therapy strategy for breast cancer.
International journal of clinical and experimental pathology. 01/2014; 7(6):2850-60.
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to investigate the permeability of exendin-4-loaded chitosan nanoparticles using the Madin Darby canine kidney (MDCK) cell monolayer as an in vitro model and the rat intestine as an ex vivo model of the human intestinal barrier. A series of formulations of sodium tripolyphosphate (TPP) and chitosan with different molecular weights and degrees of deacetylation was evaluated. The formulation consisting of 0.1% TPP and 0.2% chitosan (400 kDa, 95% degree of deacetylation), which gave optimized monodispersed particle size (303.1±10.36 nm), zeta potential (18.37±1.15 mV) and encapsulation efficiency (38.0±2.6%), was used for further analysis. After determining their biocompatibility, the transport potential of drug-loaded chitosan nanoparticles was evaluated and compared with free exendin-4 using both MDCK cell monolayers and different rat intestinal segments. Mechanisms underlying enhanced transport of exendin-4 in the cell model were also explored. Compared with free exendin-4, the absorption of optimized chitosan nanoparticles was enhanced by 4.7-fold in MDCK cell monolayers and by 2.0-2.78-fold in different rat intestinal segments, with no significant difference between the duodenum, jejunum and ileum. As supported by confocal laser scanning microscopic analysis, the lower enhancement of absorption in the intestine compared to the cell monolayer likely resulted from the chitosan nanoparticle-mediated opening of cellular tight junctions and not through intracellular transport. These findings suggest that the potential application of chitosan nanoparticles as delivery carriers of exendin-4 is limited and may need further modifications.
[Show abstract][Hide abstract] ABSTRACT: Mucin 1 (MUC1) is a tumor-associated antigen that is overexpressed in several adenocarcinomas. However, clinical trials with MUC1 showed that MUC1 is a relatively poor immunogen in humans. In view of the low immunogenicity of this protein vaccine, we designed a method based on an immunoadjuvant and immunization strategy to enhance the cellular immune response to this protein vaccine. DDA/MPL has been evaluated as an adjuvant to induce strong immunity for the tuberculosis vaccine. However, its adjuvant role combined with the vaccine targeting MUC1 in malignant carcinomas has not previously been reported. Our previous study showed that adenovirus prime protein boost vaccination could significantly enhance the cellular immunity and antitumor efficacy. In our study, we used MUC1 VNTRs as the target of cancer vaccine and DDA/MPL as the adjuvant to enhancing the cellular immunity of recombinant MUC1 protein vaccine, and an AD-9M adenoviral vector prime-recombinant protein and DDA/MPL boost (designated MUC-1 VPP vaccine) strategy was studied to enhance the antitumor efficacy. The results demonstrated that antigen-specific IFN-γ-secreting T cells were increased by 2-fold, and cytotoxic T lymphocytes (CTLs) were induced effectively when the protein vaccine was combined with the DDA/MPL adjuvant. Moreover, the vaccination induced nearly 60% inhibition of the growth of B16 melanoma in mice and prolonged the survival of tumor-bearing mice. The inhibition was correlated with the specific immune responses induced by the MUC1 VPP vaccine. The data suggested that DDA/MPL-adjuvant MUC-1 VPP vaccine may be developed into effective tumor vaccines for melanomas and possibly for other tumors expressing MUC1 protein.
[Show abstract][Hide abstract] ABSTRACT: Rabies virus (RABV) causes a fatal infectious disease, but effective protection may be achieved with the use of rabies immunoglobulin and a rabies vaccine. Virus-neutralizing antibodies (VNA), which play an important role in the prevention of rabies, are commonly evaluated by the RABV neutralizing test. For determining serum VNA levels or virus titers during the RABV vaccine manufacturing process, reliability of the assay method is highly important and mainly dependent on the diagnostic antibody. Most diagnostic antibodies are monoclonal antibodies (mAbs) made from hybridoma cell lines and are costly and time consuming to prepare. Thus, production of a cost-effective mAb for determining rabies VNA levels or RABV titers is needed. In this report, we describe the prokaryotic production of a RABV-specific single-chain variable fragment (scFv) protein with a His-tag (scFv98H) from a previously constructed plasmid in a bioreactor, including the purification and refolding process as well as the functional testing of the protein. The antigen-specific binding characteristics, affinity, and relative affinity of the purified protein were tested. The scFv98H antibody was compared with a commercial RABV nucleoprotein mAb for assaying the VNA level of anti-rabies serum samples from different sources or testing the growth kinetics of RABV strains for vaccine manufactured in China. The results indicated that scFv98H may be used as a novel diagnostic tool to assay VNA levels or virus titers and may be used as an alternative for the diagnostic antibody presently employed for these purposes.
Applied Microbiology and Biotechnology 11/2013; · 3.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although the existence of cancer stem cells (CSCs) has been demonstrated in colorectal cancer, further investigation is hindered by controversies over their surface markers. The sphere formation assay is widely used as in vitro method for derivation and characterization of CSCs based on the intrinsic self-renewal property of these cells. Isolated cancer cells that form tumorspheres are generally recognized as CSCs with self-renewal and tumorigenic capacities. In this study, colon spheres grown from Caco-2 cells in the sphere formation assay were separated from other differentiated cells and characterized. Compared with Caco-2 cells, the derived colon spheres lost several CSC properties. The colon spheres contained decreased levels of specific colorectal CSC surface markers as well as low levels of ATP-binding cassette (ABC) transporters typically overexpressed in CSCs, resulting in the near loss of their chemoresistance ability. Furthermore, cells that developed as colon spheres with strong self-renewal ability in vitro lost their tumorigenic capacity in vivo compared with Caco-2 cells, which could establish tumors in non-obese diabetic/severe-combined immunodeficient (NOD/SCID) mice. The results indicated that the Caco-2 cell derived colon spheres did not consist of colorectal CSCs. Thus, the well-accepted sphere formation assay may not be an effective method for CSC isolation and characterization from the Caco-2 colorectal cancer cell line.
Current Stem Cell Research & Therapy 11/2013; · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The HIV-1 accessory factor Vif is necessary for efficient viral infection in non-permissive cells. Vif antagonizes the antiviral activity of human cytidine deaminase APOBEC3 proteins that confer the non-permissive phenotype by tethering them (APOBEC3DE/3F/3G) to the Vif-CBF-beta-ElonginB-ElonginC-Cullin5-Rbx (Vif-CBF-beta-EloB-EloC-Cul5-Rbx) E3 complex to induce their proteasomal degradation. EloB and EloC were initially reported as positive regulatory subunits of the Elongin (SIII) complex. Thereafter, EloB and EloC were found to be components of Cul-E3 complexes, contributing to proteasomal degradation of specific substrates. CBF-beta is a newly identified key regulator of Vif function, and more information is needed to further clarify its regulatory mechanism. Here, we comprehensively investigated the functions of EloB (together with EloC) in the Vif-CBF-beta-Cul5 E3 ligase complex.
The results revealed that: (1) EloB (and EloC) positively affected the recruitment of CBF-beta to Vif. Both knockdown of endogenous EloB and over-expression of its mutant with a 34-residue deletion in the COOH-terminal tail (EloBDeltaC34/EBDeltaC34) impaired the Vif-CBF-beta interaction. (2) Introduction of both the Vif SLQ [rightwards arrow] AAA mutant (VifDeltaSLQ, which dramatically impairs Vif-EloB-EloC binding) and the Vif PPL [rightwards arrow] AAA mutant (VifDeltaPPL, which is thought to reduce Vif-EloB binding) could reduce CBF-beta binding. (3) EloB-EloC but not CBF-beta could greatly enhance the folding of full-length Vif in Escherichia coli. (4) The over-expression of EloB or the N-terminal ubiquitin-like (UbL) domain of EloB could significantly improve the stability of Vif/VifDeltaSLQ/VifDeltaPPL through the region between residues 9 and 14.
Our results indicate that the Vif interaction with EloB-EloC may contribute to recruitment of CBF-beta to Vif, demonstrating that the EloB C-teminus may play a role in improving Vif function and that the over-expression of EloB results in Vif stabilization.
[Show abstract][Hide abstract] ABSTRACT: Human enterovirus type 71 (EV71) and Coxsackievirus A group type 16 (CA16) belong to human Enterovirus species A of the family Picornaviridae. These viruses are recognized as the major pathogens responsible for epidemics of hand-foot-mouth disease (HFMD), which presents with fever and vesicular eruptions of palms, soles of the feet or mouth. Human scavenger receptor class B, member 2 (SCARB2) has been identified as the receptor for both EV71 and CA16, as overexpression of SCARB2 in cells can enhance virus replication significantly.
In this study, we used a lentivirus packaging vector to transduce the SCARB2 gene into human embryonic kidney cells (293), human rhabdomyosarcoma cells (RD) and African green monkey kidney cells (Vero) to create stable expression lines. Expression of SCARB2 in the resulting three transgenic cell lines was confirmed by real-time RT-PCR, immunofluorescence and flow cytometry.
Levels of SCARB2 mRNA determined by real-time RT-PCR in 293-SCARB2 (293S) or RD-SCARB2 (RDS) transgenic cell lines were approximately 2 x 102 times higher than those in 293 and RD cells, respectively, and three times higher in Vero-SCARB2 (VeroS) than in Vero cells. Furthermore, EV71 and CA16 virus titers in 293S and RDS cells were 102--103-fold higher (detected in RD cell) than those in the parental cells, and a 10-fold higher titer of EV71 was achieved in VeroS cells compared with that in Vero cells.
We established for the first time three cell lines stably overexpressing SCARB2, which showed drastic increases in susceptibility to EV71/CA16 infection. These optimal cell lines may be utilized to develop inactivated vaccines for EV71/CA16 and facilitate rapid detection and isolation of HFMD pathogens or other Enterovirus serotypes. Furthermore, these stable cell lines also can serve as tools to facilitate drug screenings as well as molecular studies on virus-host interactions and pathogenesis of causative agents for HFMD.
[Show abstract][Hide abstract] ABSTRACT: Tetherin/BST-2/CD317 inhibits HIV-1 release from infected cells, while HIV-1 Vpu efficiently antagonizes tetherin based on intermolecular interactions between the transmembrane domains of each protein. In this study, we successfully partially purified His-tagged tetherin with a glycophosphatidylinositol deletion (delGPI) and His-tagged full-length Vpu from transiently transfected 293T cells using affinity chromatography. The in vitro interaction between these purified proteins was observed by a pull-down assay and ELISA. Detection of the Vpu/tetherin interaction by ELISA is a novel approach that would be advantageous for inhibitor screening in vitro. Successful co-purification of the tetherin/Vpu complex also provides a basis for further structural studies.
Protein Expression and Purification 08/2013; · 1.43 Impact Factor