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Abstract
ObjectiveStudying the methylation status of long terminal repeats (LTR) and its relationship to gag expression of HIV-1 in order to explore regulation mechanism of HIV-1 gene expression in vertical transmission from sperm to embryo.Methods/Principal FindingsSperm samples were collected from a healthy donor and seven patients with HIV/AIDS. Zona-free hamster ova were fertilized by donor’s spermatozoa transfected with pIRES2-EGFP-LTR-gag and patient’s spermatozoa to obtain zygotes and 2-cell embryos, respectively. Interspecific in vitro fertilization, bisulfite sequencing PCR (BSP), RT-PCR, nested RT-PCR, nested real-time qRT-PCR and 2−△△Ct method, indirect immunofluoresence (IF) assay were performed. For donor’s samples, the methylation rates of HIV-1 LTR were 0.56%, 1.67%, 0.56%, 0.56% in plasmid, spermatozoa, zygotes and 2-cell embryos, respectively while spermatozoa were transfected with unmethylated plasmid, and were 95.0%, 84.44%, 3.3%, 1.67% while transfected with methylated plasmid. The positive bands for HIV-1 gag cDNA were detected in spermatozoa and 2-cell embryos. The positive signals for HIV-1 p24 Gag protein were detected in 2-cell embryos but not in spermatozoa. For patient’s samples, methylation rates of HIV-1 LTR were different in spermatozoa among patients. After fertilization, CpG sites in HIV-1 LTR were highly demethylated in zygotes and 2-cell embryos. The gag transcription levels increased with decreasing of methylation rates of HIV-1 LTR, which showed a strong negative correlations between gag transcription levels and methylation rates of HIV-LTR ether in the spermatozoa (r = −0.9877, P<0.0001) or in the sperm-derived 2-cell embryos (r = −0.9092, P = 0.0045).ConclusionLTR methylation regulates expression of HIV-1 gag in vertical transmission from sperm to embryo.
Highlights
Human immunodeficiency virus type 1 (HIV-1) infection is characterized by a prolonged period of latency, which may be followed by AIDS or AIDS-related complex [1]
A 2292-bp fragment was obtained by VspI and EcoRI digestion, and amplified from the constructed plasmid using a primer pair specific for long terminal repeats (LTR)-gag, which identity with that amplified from the backbone plasmid was 99.64%, indicating that the fragment of HIV-1 LTR-gag DNA has been successfully cloned into the constructed plasmid
In the second round of nested RT-PCR, the product of the first round was used as template, and a 730 bp positive band for HIV-1 P24 gag cDNA was observed in the 2-cell embryos and positive control, and no positive band was detected in the negative controls (-RT, -T)
Summary
Human immunodeficiency virus type 1 (HIV-1) infection is characterized by a prolonged period of latency, which may be followed by AIDS or AIDS-related complex [1]. Some reported the presence of HIV1 RNA, proviral DNA or protein in ejaculated spermatozoa or maturing spermatids from HIV-1 seropositive men [6,7,8]. Thereafter, it was reported that HIV-1 proviral DNA can integrate into the genomes of human sperm and mouse oocytes which were transfected with the plasmid pIRES2-EGFP-gag, respectively. After being brought into oocyte by fertilization, it can replicate with the replication of host genome and subsequently express its protein in embryo [12]. These findings indicated the possibility of true vertical transmission of HIV-1 via spermatozoa. The mechanism for regulation of HIV-1 gene expression in spermatozoa and embryonic cells is still largely unknown
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