Multiple barriers to recombination between divergent HIV-1 variants revealed by a dual-marker recombination assay

Recombination is a major force for generating human immunodeficiency virus type 1 (HIV-1) diversity and produces numerous recombinants circulating in the human population. We previously established a cell-based system using green fluorescent protein gene (gfp) as a reporter to study the mechanisms of HIV-1 recombination. We now report an improved system capable of detecting recombination using authentic viral sequences. Frameshift mutations were introduced into the gag gene so that parental viruses do not express full-length Gag; however, recombination can generate a progeny virus that expresses a functional Gag. We demonstrate that this Gag reconstitution assay can be used to detect recombination between two group M HIV-1 variants of the same or of different subtypes. Using both gfp and gag assays, we found that, similar to group M viruses, group O viruses also recombine frequently. When recombination between a group M virus and a group O virus was examined, we found three distinct barriers for intergroup recombination. First, similar to recombination within group M viruses, intergroup recombination is affected by the identity of the dimerization initiation signal (DIS); variants with the same DIS recombined at a higher rate than those with different DIS. Second, using the gfp recombination assay, we showed that intergroup recombination occurs much less frequently than intragroup recombination, even though the gfp target sequence is identical in all viruses. Finally, Gag reconstitution between variants from different groups is further reduced compared with green fluorescent protein, indicating that sequence divergence interferes with recombination efficiency in the gag gene. Compared with identical sequences, we estimate that recombination rates are reduced by 3-fold and by 10- to 13-fold when the target regions in gag contain 91% and 72-73% sequence identities, respectively. These results show that there are at least three distinct mechanisms preventing exchange of genetic information between divergent HIV-1 variants from different groups.     

Effects of recombinant adeno-associated viral vectors on angiopoiesis and osteogenesis in cultured rabbit bone marrow stem cells via co-expressing hVEGF and hBMP genes: A preliminary study in vitro

Objective

VEGF and BMP play important roles in angiogenesis and osteogenesis. Combining these two factors may be a promising therapeutic strategy for avascular necrosis of the femoral head (ANFH).

Methods

Rabbit bone marrow-derived mesenchymal stem cells (BMSCs) were isolated and purified by density gradient centrifugation combined with attachment culture methods. The purity and characteristics of the BMSCs were detected by cell surface antigen identification. The best MOI of BMSCs transfected with rAAV was detected by fluorescent cell counting, and cell viability was determined by MTT assay. Expression of the genes of interest was detected by GFP gene expression, RT-PCR assay, and ELISA assay. The biological activities of VEGF and BMP were detected by angiogenic and osteogenic assays.

Results

The best MOI of BMSCs transfected with rAAV was 5 × 10⁴ v.g./cell. Cell growth curves showed vigorous cell viability. Expressions of the GFP, VEGF165, and BMP₇ genes were detected 1 day post-transfection and peaked 14 days post-transfection. Expression of the genes of interest was sustained over 1 month. VEGF and BMP proteins secreted from BMSCs transfected with rAAV-hVEGF₁₆₅-IRES-hBMP₇ enhanced angiogenesis and osteogenesis in vitro.

Conclusion

Recombinant adeno-associated viral vectors co-expressing the hVEGF₁₆₅ and hBMP₇ genes showed efficient gene expression ability. The VEGF₁₆₅ and BMP₇ proteins expressed from the vector have efficient biological activity in vitro.