Reactivation from latency displays HIV particle budding at plasma membrane, accompanying CD44 upregulation and recruitment
© Suyama et al; licensee BioMed Central Ltd. 2009
Received: 02 March 2009
Accepted: 13 July 2009
Published: 13 July 2009
It has been accepted that HIV buds from the cell surface in T lymphocytes, whereas in macrophages it buds into intracellular endosomes. Recent studies, on the other hand, suggest that HIV preferentially buds from the cell surface even in monocytic cells. However, most studies are based on observations in acutely infected cells and little is known about HIV budding concomitant with reactivation from latency. Such studies would provide a better understanding of a reservoir for HIV.
We observed HIV budding in latently infected T lymphocytic and monocytic cell lines following TNF-α stimulation and examined the upregulation of host factors that may be involved in particle production. Electron microscopy analysis revealed that reactivation of latently infected J1.1 cells (latently infected Jurkat cells with HIV-1) and U1 cells (latently infected U937 cells with HIV-1) displayed HIV particle budding predominantly at the plasma membrane, a morphology that is similar to particle budding in acutely infected Jurkat and U937 cells. When mRNA expression levels were quantified by qRT-PCR, we found that particle production from reactivated J1.1 and U1 cells was accompanied by CD44 upregulation. This upregulation was similarly observed when Jurkat and U937 cells were acutely infected with HIV-1 but not when just stimulated with TNF-α, suggesting that CD44 upregulation was linked with HIV production but not with cell stimulation. The molecules in endocytic pathways such as CD63 and HRS were also upregulated when U1 cells were reactivated and U937 cells were acutely infected with HIV-1. Confocal microscopy revealed that these upregulated host molecules were recruited to and accumulated at the sites where mature particles were formed at the plasma membrane.
Our study indicates that HIV particles are budded at the plasma membrane upon reactivation from latency, a morphology that is similar to particle budding in acute infection. Our data also suggest that HIV expression may lead to the upregulation of certain host cell molecules that are recruited to sites of particle assembly, possibly coordinating particle production.
It has been thought that HIV particles assemble and bud at the plasma membrane (PM) in T lymphocytes and HeLa cells, but at the endosomes in macrophages, suggesting that such endosomal targeting may be essential for HIV budding in macrophages [1–6]. However, recent studies using the inhibitors of the endocytic pathway and membrane-impermeant dyes have revealed that the PM is the primary site for HIV assembly and particle budding even in macrophages and that particles accumulate at the endosomes through endocytosis [7–9]. Nevertheless, these studies are based on observations in acutely infected cells and little is known about HIV budding concomitant with reactivation from latency. Latently infected resting T cells are known to serve as a stable reservoir for HIV during anti-retroviral therapy and to produce infectious particles upon cell reactivation. Studies on HIV production from latently infected cells upon reactivation are necessary for a better understanding of HIV pathogenesis, although some studies have indicated intracellular accumulation of particles in chronically or latently infected cells [10, 11]. Here, we employed J1.1 cells that were Jurkat T lymphocytic cells latently infected with HIV-1, and U1 cells that were U937 monocytic cells latently infected with HIV-1, and observed HIV particle budding following reactivation.
Electron microscopy was carried out to examine where particle budding occurred in J1.1 and U1 cells upon reactivation (Fig. 1B). Little or no particles were produced in either cell line before TNF-α stimulation (Fig. 1B, most left panels), consistent with previous reports [11–14]. Upon stimulation, nascent budding particles were visible on the surface of nearly all J1.1 cells, similar to the case with U1 cells (Fig. 1B, arrowheads). Unexpectedly, particles in intracellular vesicles were rarely seen in both J1.1 and U1 cells (Fig. 1B, arrow). The findings were confirmed by immunoelectric microscopy using anti-HIV-1 p17MA antibody (Fig. 1B, most right panels). Next, their parental cell lines, Jurkat and U937 cells, were infected with HIV-1, and particle production in acute infection were examined by electron microscopy. Particle budding was observed predominantly at the PM of both Jurkat and U937 cells (Fig. 1C, arrowheads) but some U937 cells displayed budding into intracellular compartments (Fig. 1C, arrows). Immunoelectric microscopy indicated similar results (Fig. 1C, most right panels). For quantification, we counted the number of cells containing particles at the PM alone or that of cells containing particles at both intracellular vesicles and the PM (Fig. 1D). Budding at the PM was prominent, regardless of whether cells were acutely or latently infected, or T lymphocytic or monocytic, suggesting that unlike chronically infected cells , HIV particles are most likely budded from the PM in latently infected cells, although it cannot be ruled out in this experiment that the particles observed in extracellular spaces might be released by exocytosis.
To understand the significance of the upregulation of host molecules observed here, we examined intracellular localization of the molecules by immunostaining. No CD44 staining was found in unstimulated J1.1 and U1 cells, consistent with previous reports indicating CD44 downregulation during latency [30, 32]. Following reactivation, CD44 was visible and colocalized with the p17MA antigens at the PM. Similarly, CD63 and HRS stainings were rarely seen in unstimulated cells but became visible and colocalized with the 17MA signals, especially in U1 cells (Fig. 3B). These findings were very apparent in acutely infected cells (Fig. 3C): CD63 recruitment, as reported previously for acutely infected Jurkat T cells and macrophages [7, 25, 33, 34], and HRS and CD44 accumulations to the sites where mature particles were formed. Together, our data suggest that HIV expression may lead to the upregulation of certain host molecules that are recruited to the sites of particle assembly, possibly to coordinate particle production. Because CD44 is a cell adhesion molecule that mediates lymphocyte aggregation and homing [35, 36], it is conceivable that the CD44 recruitment to HIV assembly sites may lead to an efficient cell-to-cell transmission of HIV and infected cell migration to lymph nodes.
In conclusion, despite numerous literature on HIV budding to intracellular compartments especially in macrophages, our data indicate that upon reactivation from latent infection, HIV predominantly buds at the PM, a morphology that is similar to particle budding in acute infection, suggesting that HIV latency have a potential for robust production of HIV observed for acute infection.
human immunodeficiency virus
tumor necrosis factor-α
tumor susceptibility gene-101
hepatocyte growth factor regulated tyrosine kinase substrate
early endosomal antigen 1
synaptosome associated 23 kDa protein.
We thank M. Nakamura (Kitasato University, Japan) and Y. Fujioka (Osaka Medical College, Japan) for supporting electron microscopy analysis. This work was supported by an AIDS grant from the Ministry of Health, Labor, and Welfare of Japan and a grant from the Ministry of Sciences, Sports, and Education of Japan.
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