Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos

PLOS ONE, Dec 2019

Objective Studying 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 Findings Sperm 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). Conclusion LTR methylation regulates expression of HIV-1 gag in vertical transmission from sperm to embryo.

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Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos

et al. (2013) Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm- Derived Embryos. PLoS ONE 8(1): e54801. doi:10.1371/journal.pone.0054801 Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm- Derived Embryos FangZheng Li 0 LianBing Li 0 Ying Zhong 0 QingDong Xie 0 JiHua Huang 0 XiangJin Kang 0 Dian Wang 0 Lan Xu 0 TianHua Huang 0 K.T. Jeang, National Institute of Health, United States of America 0 1 Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Research Center for Reproductive Medicine, Shantou University Medical College , Shantou, Guangdong , China , 2 Key Laboratory of Birth Defects and Reproductive Health , Chongqing , China , 3 Center for Reproductive Medicine, Chengdu Jinjiang Hospital for Maternal and Child Health Care , Chengdu, Sichuan , China Objective: Studying 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 Findings: Sperm samples were collected from a healthy donor and seven patients with HIV/AIDS. Zonafree 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 22ggCt 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 = 20.9877, P,0.0001) or in the spermderived 2-cell embryos (r = 20.9092, P = 0.0045). Conclusion: LTR methylation regulates expression of HIV-1 gag in vertical transmission from sperm to embryo. - Funding: This work was financially supported by the National Natural Science Foundation of China and the Li Ka Shing Shantou University Foundation. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 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]. The dramatic spread of HIV/AIDS worldwide represents a serious menace to human health, survival and social development. Understanding of the routes of HIV-1 transmission and its mechanism is an important prerequisite for controlling the HIV/AIDS pandemic. The typical routes of HIV-1 transmission are sexual intercourse, blood-to-blood contact, and perinatal transmission from motherto-child [25]. In the recent years, the true vertical transmission of HIV-1 via germ line, a new but not yet fully proven route, has attracted plenty of attention. Some reported the presence of HIV 1 RNA, proviral DNA or protein in ejaculated spermatozoa or maturing spermatids from HIV-1 seropositive men [68]. Baccetti et al. observed that HIV-1 can bind to and enter normal sperm, and the viral particles, their nucleic acids and antigens were present in the cytoplasm of sperm from HIV-1 infected men, and such sperm can transfer HIV-1 like particles to normal human oocytes through in vitro fertilization [9]. 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. The sperm- or oocyteintroduced HIV-1 gag genes retained their functions in replication, transcription and translation in the embryonic cells [10,11]. More recently, Wang et al. found that HIV-1 proviral sequences were able to integrate into sperm nuclei and chromosomes of HIV/AIDS patients. 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. However, the mechan (...truncated)


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FangZheng Li, LianBing Li, Ying Zhong, QingDong Xie, JiHua Huang, XiangJin Kang, Dian Wang, Lan Xu, TianHua Huang. Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos, PLOS ONE, 2013, 1, DOI: 10.1371/journal.pone.0054801