Experimental Study On Vertical Transmission Of HIV-1 Gag Gene Via Oocyte

【BACKGROUND & AIM】Since 1983, when the human immunodeficiency virus (HIV) was first identified, there has been a dramatic spread of this virus. It is generally believed that suppression of the immune response, either by the virus or by other means, is a predisposing factor. Bagasra et al found that HIV-1 proviral DNA sequences could be detected in sperm of a significant percentage of HIV-1 infected men (45%) at all stages of clinical immunodeficiency. Using immunocytochemistry and in situ hybridization at the electron microscope level, and the PCR technique, Baccetti et al demonstrated that HIV-1 can bind to and enter normal sperm. This study also showed that viral particles, their antigens, and nucleic acid were present in sperm from HIV-1 infected men, and that these sperm could transfer HIV-1 like particles to normal human oocytes. This indicates that true vertical transmission of HIV via germ cells may actually occur. To confirm this, it must first be established that HIV-1 genes can integrate into the genome of host germ cells. Secondly, sperm- or oocyte-mediated HIV genes must be able to be replicated and to be functionally expressed in embryonic cells.Since the HIV-1 gag gene is needed to make the structural proteins for new virus particles, it plays an important role in HIV pathogenesis. The current study therefore addresses aspects of the integration of HIV-1 gag gene into the mouse oocyte genome. It also examines further replication of this gene, as well as its transcription and translation in embryonic cells, to provide further experimental evidence for vertical transmission of HIV-1 gag gene via female germ line.【MATERIALS AND METHODS】Recombinant plasmids pIRES2-EGFP-gag were constructed in our laboratory. The feasibility of vertical transmission of HIV-1 gag gene via oocytes was explored by injecting pIRES2-EGFP-gag plasmids into mouse ovaries to transfect germ cells. Induction of superovulation was then used to produce oocytes, and animal mating was used to produce two-cell embryos. PCR and Fluorescence in situ hybridization (FISH) were performed to detect the intergration of HIV-1 gag DNA in nucleus and chromosomes of oocyte, and to detect the replication of HIV-1 gag gene in embryonic cells. RT-PCR was performed to detect transcription of HIV-1 gag gene in oocytes, and in embryonic cells. Immunofluorescence assay was performed to detect translation of HIV-1 gag gene in oocytes and in embryonic cells.【RESULTS】①Recombinant plasmid pIRES2-EGFP-gag containing the provirus DNA of HIV-1 gag and EGFP genes expressed EGFP as reporter for gag gene expression, which made it possible to distinguish between oocytes or between the embryos, which carrying HIV-1 gag gene or not, by the presence of green fluorescence.②PCR detected the positive bands for HIV-1 gag gene in the tested oocytes with green fluorescence.③FISH revealed that the positive signals for HIV-1 gag DNA were presented in nucleus of immature oocyte and in chromosomes of mature oocytes, and in each nucleus of 2-cell embryos.④RT-PCR showed the specific bands for HIV-1 gag cDNA in immature and mature oocytes as well as in two-cell embryos.⑤Immunofluoresence assay presented the positive signals for HIV-1 p24 gag protein within cytoplasm of immature and mature oocytes as well as in two-cell embryos.【CONCLUSION】①The application of pIRES2-EGFP-gag plasmid made it possible to determine which oocyte or embryo contain virus gene by observation of green fluorescence. This was not only less time consuming and less expensive but also the results were more accurate and reliable.②HIV-1 gag gene was able to pass through zona pellucida and plasma membrane of oocyte and intergrate into host genome.③Oocyte carrying HIV-1 gag gene was able to achieve normal fertilization.④The replication of oocyte-mediated HIV-1 gag gene was synchronize with that of the host genome, and then the two gene copies were segregated into the two daughter cells during the first cleavage.⑤HIV-1 gag gene integrated into oocyte genome or introduced into embryo was able to be transcribed into mRNA in immature and mature oocytes as well as in embryonic cells.⑥HIV-1 gag gene integrated into oocyte genome or introduced into embryo was able to be translated to make HIV-1 p24 gag protein in immature and mature oocytes as well as in embryonic cells.⑦Our results provided the solid evidences that HIV-1 gag gene are able to transmit vertically to next generation via female germ line.⑧To our knowledge, this is the first systematic investigation of the vertical transmission of the HIV-1 gag gene via oocytes.