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46
HIV Assembly in, and Release from, Primary Macrophages
Mark Marsh*, A Pelchen-Matthews, B Kramer, R Byland, M Deneka, and A Fraile-Ramos
Univ Coll, London, UK
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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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