11th Conference on Retroviruses and Opportunistic Infections San Francisco, California - February 8 - 11, 2004

Conf Retrovir Opportunistic Infect 2004 Feb 8-11;11:abstract no. 46

Mark Marsh, A Pelchen-Matthews, B Kramer, R Byland, M Deneka, and A Fraile-Ramos
Univ Coll, London, UK

BACKGROUND: HIV types 1 and 2, and SIV, are generally thought to assemble at the plasma membrane of infected cells.

METHODS: We investigated virus assembly by immunolabeling cryosections of HIV-1-infected primary human monocyte-derived macrophages (MDM). Using fluorescence and electron microscopy we found virus particles, identified by their morphology and positive labelling with antibodies to the viral p17, p24, and envelope proteins, in intracellular vacuoles.

RESULTS: These compartments were strongly positive for the late endosomal marker CD63. CD63 was enriched on vesicles within these structures and was seen to be incorporated into the envelopes of budding and mature virions in these organelles. The virus-containing vacuoles were also labeled with antibodies against LAMP-1, CD81, and CD82, and these antigens were seen to be incorporated into the viral envelope. By these criteria we identified the intracellular virus-containing vesicles as late endosomes. Profiles of budding virions were for the most part seen in late endosomes and not at the cell surface in these cells. To assess the cellular source of infectious viruses derived from MDM, virus-containing media from infected cells were precipitated with specific antibodies. Only antibodies directed against antigens found in late endosomes precipitated infectious virus, while antibodies against proteins located primarily on the cell surface did not. Our data indicate that the majority of the infectious HIV produced by primary macrophages is assembled on late endocytic membranes and acquires antigens characteristic of this compartment. Moreover, we also find that the human cytomegalovirus (HCMV) undergoes its final assembly events on CD63 positive membranes, including late endosomes, and that extracellular HCMV contains CD63 in its membrane.

CONCLUSIONS: Virus release appears to occur by fusion of virus-containing vesicles with the plasma membrane. Currently, the molecular mechanisms underlying these secretory events are not understood. We suggest that assembly on endocytic membranes is not unique to HIV and that the specific location of this assembly, on compartments that in many cells have secretory functions, has significant implications for the biology of these viruses in vivo and for their cell-to-cell transmission.

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