Abstract
Immune progenitor cells differentiate in bone marrow (BM) and then migrate to tissues. HIV-1 infects multiple BM cell types, but virus dissemination within BM has been poorly understood. We used light microscopy and electron tomography to elucidate mechanisms of HIV-1 dissemination within BM of HIV-1-infected BM/liver/thymus (BLT) mice. Tissue clearing combined with confocal and light sheet fluorescence microscopy revealed distinct populations of HIV-1 p24-producing cells in BM early after infection, and quantification of these populations identified macrophages as the principal subset of virus-producing cells in BM over time. Electron tomography demonstrated three modes of HIV-1 dissemination in BM: (i) semi-synchronous budding from T-cell and macrophage membranes, (ii) mature virus association with virus-producing T-cell uropods contacting putative target cells, and (iii) macrophages engulfing HIV-1-producing T-cells and producing virus within enclosed intracellular compartments that fused to invaginations with access to the extracellular space. These results illustrate mechanisms by which the specialized environment of the BM can promote virus spread locally and to distant lymphoid tissues.
Highlights
The rapid systemic dissemination of HIV-1 from the site of initial infection to distant lymphoid tissues is a critical component of the acute phase of HIV-1 infection
In direct comparisons of electron tomography (ET) imaging of Bone marrow (BM) obtained by needle aspiration versus after dissection and removal from long bones, we found that BM extracted by needle aspiration was contaminated with blood from the vasculature, and we could only obtain physiologically-relevant regions of BM that were distinct from contaminating mature red blood cells at the electron microscopy (EM) level by extracting BM samples from long bones
Light and electron microscopy of HIV-1–infected cultured cells have revealed important insights into the HIV-1 life cycle and mechanisms of virus transmission (Bennett et al, 2009; Bracq et al, 2017; Deneka et al, 2007; Folks et al, 1988; Martin et al, 2010; Sougrat et al, 2007), but cultured cells cannot replicate complex interactions encountered in vivo by viruses in the context of functioning immune cell populations within tissues
Summary
The rapid systemic dissemination of HIV-1 from the site of initial infection to distant lymphoid tissues is a critical component of the acute phase of HIV-1 infection. To address mechanisms of HIV-1 dissemination in BM, we employed a multiscale imaging approach: Bone CLARITY (Greenbaum et al, 2017) combined with IF, confocal, and LSFM to quantify the distributions of specific cell types associated with HIV-1 within intact volumes of BM; and ET to detect individual virions and virion-producing cells to characterize aspects of HIV-1 infection at the subcellular level. These imaging studies allowed visualization of HIV-1 distribution within larger volumes of tissue from a hu-mouse model and revealed distinct mechanisms of virus dissemination within BM
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