Feline calicivirus capsid protein expression and capsid assembly in cultured feline cells

The capsid protein of feline calicivirus (FCV) was expressed by using plasmids containing cytomegalovirus, simian virus 40, or T7 promoters. The strongest expression was achieved with the T7 promoter and coinfection with vaccinia virus expressing the T7 RNA polymerase (MVA/T7pol). The FCV precursor capsid protein was processed to the mature-size protein, and these proteins were assembled in to virus-like particles.     

Expression of HIV-1 epitopes included in particles formed by human hepatitis B virus nucleocapsid protein

Hybrids of the core protein of hepatitis B virus (HBcAg) have been designed which carry N-terminal insertions of B- and T-cell epitopes of HIV-1 an immunodominant B-epitope from gp41, a T-cell epitope from p34 pol, and a cluster of B- and T-cell epitopes from p17 gag. The hybrids have been synthesized using two expression systems-one based on the thermoinducible PR promoter of bacteriophage lambda and the other one based on phi 10 promoter of bacteriophage T7 with 3-5% and 7-14% yields, respectively. The hybrids have dual HBV and HIV-1 immunospecificity and are assembled into particles similar to those formed by the protein carrier HBcAg. Sandwich ELISA and immune electron microscopy revealed that HIV-1 epitopes are exposed on the surface of the particles.     

Factors influencing pulmonary surfactant protein A levels in cord blood, maternal blood and amniotic fluid

The sequence encoding the truncated core protein (amino acids 1-98) of hepatitis C virus (HCc) was expressed in E. coli for production of HCc(1-98), or fused with the truncated core antigen (HBcAg) and segments from the preS1 and preS2 regions from hepatitis B virus (HBV) for production of HBcPreS1PreS2HCc(1-98). The HCc(1-98) and HBcPreS1PreS2HCc(1-98) proteins reacted with sera from HCV-infected individuals by immunoblot analyses, while the latter protein also exhibited HBV core antigenicity. They induced antibodies against HBcAg and/or HCV core protein in rabbits and in mice. Moreover, HBcPreS1PreS2HCc(1-98) is more immunogenic than HCc(1-98) in terms of anti-HCc induction. An ELISA that employed recombinant HCV core antigens of either HCc(1-98) or HBcPreS1PreS2HCc(1-98) to detect anti-HCc and/or anti-HBc antibodies was developed. Evaluation of serum samples with different status of HBV and HCV infections suggested that HCc(1-98) might be suitable for the determination of antibodies against HCV core protein, while HBcPreS1PreS2HCc(1-98) might be of value to detect HCV and/or HBV infection in donated blood in HBV low-prevalence countries.     

Production of uninfectious human immunodeficiency virus type 1 containing viral protein R fused to a single-chain antibody against viral integrase

A single-chain antibody (scAb) against human immunodeficiency virus type 1 (HIV-1) integrase was expressed as a fusion protein of scAb and HIV-1 viral protein R (Vpr), together with the HIV-1 genome, in human 293T cells. The expression did not affect virion production much but markedly reduced the infectivity of progeny virions. The fusion protein was found to be incorporated into the virions. The incorporation appears to account for the reduced infectivity.     

Murine retroviral vector that induces long-term expression of HIV-1 envelope protein

A retroviral vector was constructed that induces long-term expression of human immunodeficiency virus type 1 (HIV-1) rev, vpu and env genes. The vector contains the neo gene and a cytomegalovirus (CMV) immediate early promoter followed by HIV-1 sequence. When HeLa cells were infected with viral stocks derived from this vector, about 25% of the resulting G418-resistant clones expressed HIV-1 envelope protein (Env), easily detectable by Western blot analysis, metabolic labelling, and syncytium formation after co-cultivation with HeLa-CD4 cells. In most cases the level of Env expression was higher than in a T cell line (H9) chronically infected with HIV-1. Env-expressing HeLa cell lines also expressed Rev, detected by transfection with a Rev-dependent CAT gene construct, and Vpu, detected by immunoprecipitation with a Vpu-specific antiserum. The 75% of G418-resistant HeLa cell lines that did not express Env were found to contain proviruses that had undergone deletion of env sequences corresponding to a known intron; presumably these cell lines arose as a result of infection with virions derived from spliced RNAs. This vector should be useful for studying non-transient effects of HIV Env, Rev and Vpu in tissue culture, and for the production of Env- and/or Rev-expressing cell lines.     

Analysis of promoters in Borrelia burgdorferi by use of a transiently expressed reporter gene

A transient chloramphenicol acetyltransferase (CAT) expression system was developed for Borrelia burgdorferi. An Escherichia coli vector containing a promoterless Streptococcus agalactiae cat gene was constructed. Promoters for ospA, ospC, and flaB were placed upstream of this cat gene, and CAT assays were performed in E. coli from these stably maintained plasmids. The plasmids with putative promoters ospA and flaB were found to be approximately 20-fold more active than were the plasmids with ospC or no promoter. The level of activity correlated well with the resistance to chloramphenicol that each plasmid provided. Next, the nonreplicative plasmid constructs were transformed by electroporation into B. burgdorferi. CAT assays were performed by both thin-layer chromatography and the fluor diffusion method. Measurement of CAT activity demonstrated that the ospA promoter was again about 20-fold more active than the promoterless cat gene. The flaB and ospC promoters increased the activity seven- and threefold, respectively, over that with the promoterless construct. This simple transient-expression assay was shown to be an effective method to study promoter function in B. burgdorferi in the absence of a well-developed genetic system.