期刊
NATURE METHODS
卷 5, 期 1, 页码 75-85出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH1137
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资金
- NIAID NIH HHS [AI040877, AI065412, R37 AI040877, AI065413, AI052048] Funding Source: Medline
- NICHD NIH HHS [HD041752] Funding Source: Medline
- NIDCR NIH HHS [DE015512] Funding Source: Medline
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [P01AI052048, R01AI040877, R37AI040877, U19AI065413, U19AI065412] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF DENTAL &CRANIOFACIAL RESEARCH [R01DE015512] Funding Source: NIH RePORTER
The challenge in observing de novo virus production in human immunodeficiency virus (HIV)-infected dendritic cells (DCs) is the lack of resolution between cytosolic immature and endocytic mature HIV gag protein. To track HIV production, we developed an infectious HIV construct bearing a diothiol-resistant tetracysteine motif (dTCM) at the C terminus of HIV p17 matrix within the HIV gag protein. Using this construct in combination with biarsenical dyes, we observed restricted staining of the dTCM to de novo -synthesized uncleaved gag in the DC cytosol. Co-staining with HIV gag antibodies, reactive to either p17 matrix or p24 capsid, preferentially stained mature virions and thus allowed us to track the virus at distinct stages of its life cycle within DCs and upon transfer to neighboring DCs or T cells. Thus, in staining HIV gag with biarsenical dye system in situ, we characterized a replication-competent virus capable of being tracked preferentially within infected leukocytes and observed in detail the dynamic nature of the HIV production and transfer in primary DCs.
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