Cell-to-Cell Transmission Can Overcome Multiple Donor and Target Cell Barriers Imposed on Cell-Free HIV

et al. (2013) Cell-to-Cell Transmission Can Overcome Multiple Donor and Target Cell Barriers Imposed on Cell-Free HIV. PLoS ONE 8(1): e53138. doi:10.1371/journal.pone.0053138 Cell-to-Cell Transmission Can Overcome Multiple Donor and Target Cell Barriers Imposed on Cell-Free HIV Peng Zhong 0 Luis M. Agosto 0 Anna Ilinskaya 0 Batsukh Dorjbal 0 Rosaline Truong 0 David Derse 0 Pradeep D. Uchil 0 Gisela Heidecker 0 Walther Mothes 0 Fabrizio Mammano, INSERM, France 0 1 Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America, 2 HIV Drug Resistance Program, National Cancer Institute-Frederick , Frederick, Maryland , United States of America Virus transmission can occur either by a cell-free mode through the extracellular space or by cell-to-cell transmission involving direct cell-to-cell contact. The factors that determine whether a virus spreads by either pathway are poorly understood. Here, we assessed the relative contribution of cell-free and cell-to-cell transmission to the spreading of the human immunodeficiency virus (HIV). We demonstrate that HIV can spread by a cell-free pathway if all the steps of the viral replication cycle are efficiently supported in highly permissive cells. However, when the cell-free path was systematically hindered at various steps, HIV transmission became contact-dependent. Cell-to-cell transmission overcame barriers introduced in the donor cell at the level of gene expression and surface retention by the restriction factor tetherin. Moreover, neutralizing antibodies that efficiently inhibit cell-free HIV were less effective against cell-to-cell transmitted virus. HIV cell-to-cell transmission also efficiently infected target T cells that were relatively poorly susceptible to cell-free HIV. Importantly, we demonstrate that the donor and target cell types influence critically the extent by which cell-to-cell transmission can overcome each barrier. Mechanistically, cell-to-cell transmission promoted HIV spread to more cells and infected target cells with a higher proviral content than observed for cell-free virus. Our data demonstrate that the frequently observed contact-dependent spread of HIV is the result of specific features in donor and target cell types, thus offering an explanation for conflicting reports on the extent of cell-to-cell transmission of HIV. - Funding: The work was supported by the National Institutes of Health R01s CA098727 & AI084096 to WM, and a fellowship from the China Scholarship CouncilYale World Scholars in the Biomedical Sciences to PZ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Viruses can spread either by a cell-free mode through the extracellular space or by cell-to-cell transmission through direct cell-cell contact [1,2,3,4]. For many viruses, preferences for either pathway have been known for many years. Many bacteriophages and some Alphaviruses are highly infectious in their cell-free form and a single viral particle can enter a cell and cause an infection [5,6]. If these viruses also use cell-cell contact to spread is unknown. In contrast, the infectivity to particle ratio of other viruses can be very poor despite the observation of efficient spreading in tissue cultures [7,8,9]. This observation prompted the study of cell-to-cell transmission. The inability of neutralizing antibodies that block cell-free virus to interfere with spreading of certain viruses in cultures provided early evidence for cell-to-cell spread [10,11,12,13,14]. In addition, the ability of neurotropic viruses to spread along neurons or the ability of Vaccinia virus to induce actin tails that could propel viral particles to neighboring cells supported viral spread by cell-cell contact [15,16,17,18,19]. One of the best-studied viruses is the Human immunodeficiency virus (HIV) and strong support for viral spreading by cell-to-cell transmission has accumulated over the years [1,3,4]. HIV infection of target cells via direct cell-cell contact can be 10 1000 fold more efficient than passive dissemination of virions through the extracellular milieu [8,9,20,21,22]. HIV spreading in cell culture has also been observed to be resistant to neutralizing antibodies and to the antiviral drug tenofovir, which efficiently inhibit cell-free HIV [12,20,23,24]. The current concept to explain these observations can be described by the virological synapse, a virus-induced synaptic-like contact between infected cells and uninfected target cells [23,25,26,27,28,29,30,31]. The virological synapse is believed to efficiently coordinate several steps of the viral life cycle [1,3,4]. Tight cell-cell contacts can explain why neutralizing antibodies have limited access to cell-free virus transmitted at the cell-cell interface. Cell-cell contact sites may allow for the transmission of multiple viruses generating a high local MOI [32,33], a phenomenon that has also been vividly documented in time-lapse videos monitoring multiple transmission events at cell-cell contact sites [23,34,35]. While the evidence for cell-to-cell transmission is strong and accumulating, it is not without controversy. First, in a head-tohead comparison of HIV and HTLV transmission, HIV was observed to spread mostly by a cell-free mode [36]. Second, in contrast to the higher proviral HIV content found in tissues and in co-cultures [32,33], circulating human lymphocytes were found to carry only one provirus, which might be more consistent with infections by cell-free HIV [37]. Third, conflicting observations have been reported about the ability of neutralizing antibodies to block cell-to-cell transmission [20,21,38,39]. Fourth, restriction factors such as tetherin and TRIM5 have been observed to be ineffective against infection in co-culture conditions [40,41], yet their role as restriction factors is well established [42,43,44,45,46]. Potentially, a versatile virus like HIV could use both modes of transmission to spread, but the conditions that govern either mode are poorly understood. Given the continued controversy, we followed a systematic approach to better understand the conditions that drive virus to spread by a cell-free or a cell-to-cell mode of transmission. We reasoned that any virus should be able to efficiently spread by a cell-free mode if the following criteria are met (Figure 1A): 1) Viral gene expression should be high to promote virus assembly and release from the infected donor cell, 2) once assembled, viruses should be released efficiently into the extracellular space, 3) extracellular viruses need to be stable, and 4) viruses must bind efficiently and enter target cells. Indeed, using HIV as a model virus we demonstrate that if all the above criteria are met, this virus can efficiently sp (...truncated)