Electron Tomography of HIV-1 Infection in GutAssociated Lymphoid Tissue

Critical aspects of HIV-1 infection occur in mucosal tissues, particularly in the gut, which contains large numbers of HIV-1 target cells that are depleted early in infection. We used electron tomography (ET) to image HIV-1 in gut-associated lymphoid tissue (GALT) of HIV-1-infected humanized mice, the first three-dimensional ultrastructural examination of HIV-1 infection in vivo. Human immune cells were successfully engrafted in the mice, and following infection with HIV-1, human T cells were reduced in GALT. Virions were found by ET at all stages of egress, including budding immature virions and free mature and immature viruses. Immuno-electron microscopy verified the virions were HIV-1 and showed CD4 sequestration in the endoplasmic reticulum of infected cells. Observation of HIV-1 in infected GALT tissue revealed that most HIV-1-infected cells, identified by immunolabeling and/or the presence of budding virions, were localized to intestinal crypts with pools of free virions concentrated in spaces between cells. Fewer infected cells were found in mucosal regions and the lamina propria. The preservation quality of reconstructed tissue volumes allowed details of budding virions, including structures interpreted as host-encoded scission machinery, to be resolved. Although HIV-1 virions released from infected cultured cells have been described as exclusively mature, we found pools of both immature and mature free virions within infected tissue. The pools could be classified as containing either mostly mature or mostly immature particles, and analyses of their proximities to the cell of origin supported a model of semi-synchronous waves of virion release. In addition to HIV-1 transmission by pools of free virus, we found evidence of transmission via virological synapses. Three-dimensional EM imaging of an active infection within tissue revealed important differences between cultured cell and tissue infection models and furthered the ultrastructural understanding of HIV-1 transmission within lymphoid tissue. Author Summary HIV/AIDS remains a global public health problem with over 33 million people infected worldwide. High-resolution imaging of infected tissues by three-dimensional electron microscopy can reveal details of the structure of HIV-1, the virus that causes AIDS, how it infects cells, and how and where the virus accumulates within different tissue sub-structures. Three-dimensional electron microscopy had previously only been performed to image infected cultured cells or purified virus. Here we used three-dimensional electron microscopy to examine an active infection in the gastrointestinal tract of HIV-1-infected mice with humanized immune systems, allowing visualization of the interplay between the virus and host immune cells. Recapitulating the course of infection in humans, immune cells were depleted in infected humanized mouse gut-associated lymphoid tissue, and individual HIV-1 particles were detected as they budded from host cells and accumulated in pools between cells. HIV-1 was mapped to different substructures and cell types within the gut, and free virions were found to accumulate in pools between cells and also to infect adjacent cells via regions of cell-to-cell contact called virological synapses. Our three-dimensional imaging of an HIV-1 infection in tissue uncovered differences between cultured cell and tissue models of HIV-1 infection and therefore furthered our understanding of HIV-1/AIDS as a disease of mucosal tissues.