Date Published: November 3, 2016
Publisher: Public Library of Science
Author(s): Mikaël Boullé, Thorsten G. Müller, Sabrina Dähling, Yashica Ganga, Laurelle Jackson, Deeqa Mahamed, Lance Oom, Gila Lustig, Richard A. Neher, Alex Sigal, Susan R Ross.
Cell-to-cell spread of HIV, a directed mode of viral transmission, has been observed to be more rapid than cell-free infection. However, a mechanism for earlier onset of viral gene expression in cell-to-cell spread was previously uncharacterized. Here we used time-lapse microscopy combined with automated image analysis to quantify the timing of the onset of HIV gene expression in a fluorescent reporter cell line, as well as single cell staining for infection over time in primary cells. We compared cell-to-cell spread of HIV to cell-free infection, and limited both types of transmission to a two-hour window to minimize differences due to virus transit time to the cell. The mean time to detectable onset of viral gene expression in cell-to-cell spread was accelerated by 19% in the reporter cell line and by 35% in peripheral blood mononuclear cells relative to cell-free HIV infection. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed onset. We recapitulated the earlier onset by infecting with multiple cell-free viruses per cell. Surprisingly, the acceleration in onset of viral gene expression was not explained by cooperativity between infecting virions. Instead, more rapid onset was consistent with a model where the fastest expressing virus out of the infecting virus pool sets the time for infection independently of the other co-infecting viruses.
Cell-to-cell spread of HIV is a mechanism of viral transmission whereby interaction between an infected donor cell and an infectable target cell leads to the directed transmission of virions to the target cell. Such interactions can occur between donor and target cells by various mechanisms [1–12], all of which involve the directed delivery of virions very close to the target cell, minimizing the distance over which virions need to diffuse and the consequent loss of virions en route [1–9, 11–24]. Because of the resulting high efficiency of viral delivery, target cells in cell-to-cell spread are exposed to multiple virions per cell both in in vitro infections and in vivo [17, 18, 25–31]. Multiple infections per cell decrease the sensitivity of cell-to-cell spread to antiretroviral drugs [17, 25, 27, 32, 33] and neutralizing antibodies [18, 34–36], and can overcome low infectivity and cellular restriction factors , since they increase the chances that at least one of the transmitted virions will successfully infect the cell despite inhibitors or unfavorable infection conditions [27, 38]. Because the source of insensitivity to inhibitors in cell-to-cell spread of HIV derives from multiple infections per cell, it is expected that sufficiently high inhibitor concentrations, or inhibitors more adept at suppressing multiple infections, could overcome this barrier [32, 33]. Conversely, cell-to-cell spread would offer a window of opportunity for HIV to evolve resistance to antiviral inhibitors .
We have observed faster onset of viral gene expression in coculture infection containing cell-to-cell spread of HIV relative to cell-free HIV infection. The earlier onset of viral gene expression in coculture was lost when target cells were separated from donor cells by a transwell membrane. A faster virus cycle in cell-to-cell spread relative to the non-directed, cell-free mode of infection has been previously observed directly [2, 13, 41] and inferred through modelling of infection dynamics [39, 40]. Here we used time-lapse microscopy of HIV infection to directly quantify and investigate the mechanism behind the faster onset of viral gene expression. We minimized possible differences between cell-to-cell spread and cell-free infection in the extracellular transit time from donor to target cell by limiting the time window of transmission to 2 hours. We have also minimized any contribution of virus sequence to different viral gene expression dynamics by using viruses with identical sequences derived from a molecular clone. Hence, variability in gene expression is a result of the interaction of the virus with the host cell. After exclusion of donor-target cell fusions, we found a minimum time for early viral protein expression in both infection modes, corresponding to a period of intracellular delay indicative of true infection [53–55]. We found that we could recapitulate the faster onset of viral gene expression by increasing the MOI of cell-free virus, and that there was no evidence for cooperativity or interference between co-infecting viruses. There was also no evidence for trans-acceleration of HIV gene expression onset from the surrounding infected cells.