Regulation of HIV-1 transmission by host & environmental factors
I was born and raised in Cyprus, and completed my Genetics BSc at the University of York in England. As part of my BSc, I spent a year working as a research scientist at AstraZeneca. I joined the CMB PhD program in 2009, and became a member of the Thali lab in August 2010. I have presented my work at international meetings, including two oral presentations (at the 2013 EMBO Workshop on Cell-Cell Fusion in Israel, and at the 2014 Cold Spring Harbor Retroviruses conference).
One of the main interests of the Thali group focuses on the mechanism underlying host regulation of HIV-1-induced cell-cell fusion. Tetraspanins are small transmembrane proteins considered to be membrane scaffolds, which form membrane microdomains and recruit other partner proteins, including integrins, EWI-2, and ezrin. Tetraspanins have been found (by our group and others) to repress the fusion of HIV-1 virions to target cells, reducing viral infectivity, while also repressing viral Env-dependent fusion between infected cells and uninfected target cells. Tetraspanins are enriched at viral assembly and budding sites, and incorporated into virions. Interestingly, while HIV-1 infection results in the overall downregulation of tetraspanins from the cell surface, viral Gag also specifically traps the tetraspanin CD9 at assembly sites, suggesting that the role of tetraspanins in HIV-1 infection is not merely that of a host restriction factor. In fact, the primary mode of spread of HIV-1 within the infected host is through cell-to-cell transmission, during which the fusion of infected and uninfected cell would be detrimental to viral spread, suggesting that the virus may be utilizing tetraspanins to its benefit by preventing cell-cell fusion during transmission. In support of this idea, we find that Env mutants with reduced propensity for cell-cell fusion, but no defect in viral particle release or infectivity, are able to transmit virus more efficiently than the parental wild type Env.
My recent work has primarily focused on two questions:
– What is the stage of fusion that tetraspanins regulate, and how does this affect various aspects of viral spread (especially bystander cell killing)? – In our 2014 publication in the open access journal Viruses, we presented evidence showing that tetraspanin overexpression in HIV-1 producer cells results in sensitization to fusion inhibitors targeting stages prior to (or at) formation of the hemifusion intermediate, and desensitization at late-stage inhibitors which act after opening of the fusion pore. Furthermore, using an imaging-based dye transfer assay, we found that tetraspanin overexpression also leads to accumulation of the hemifusion intermediate. Taken together, these results suggest that tetraspanins act at a stage after hemifusion but likely prior to pore opening. Intriguingly, this might allow HIV-1-infected cells to transiently fuse with uninfected target cells without progressing to an open fusion pore, which would be in line with a previously reported ability of infected cells to induce bystander cell apoptosis by transient hemifusion. We have developed live cell imaging assays to monitor hemifusion and full fusion (see figure and movie below), and have recently begun a collaboration with Dr. David Piwnica-Worms at the MD Anderson Cancer Center in Texas to introduce a fluorescent live cell apoptosis sensor in this assay and ask whether this phenomenon is facilitated by tetraspanins.
– How do environmental factors influence the behavior of HIV-1-infected cells? – Three-dimensional (3D) cell culture systems, while highly prevalent in certain fields such as cancer biology, have been largely ignored in the field of retroviruses. The behavior of infected cells is almost always studied under culture conditions which do not resemble the natural environment (i.e. within lymphoid organs) in which viral spread takes place within infected individuals. We have therefore begun developing 3D cell culture to investigate functional aspects of HIV-1 transmission, especially with regards to live cell imaging. We were very quickly able to observe phenotypes which recapitulate a previous report by Thorsten Mempel and colleagues which utilized HIV-1-infected humanized mice, including the presence of small, mobile, elongated syncytia, which are not observed in classical 2D culture (where, instead, large, round, immobile syncytia are very common). This led us to ask what aspects of 3D cell culture contribute to this dramatically different morphology, and we are in the process of investigating various biophysical, biochemical, and behavioral parameters.
Figure description: Jurkat cells were transduced with a lentiviral overexpression vector (empty or CD9), and subsequently infected with HIV pNL4-3 iGFP (Env dCT). After two days, the infected cells were co-cultured with CEM-ss cells labeled with a blue cytoplasmic dye (CMAC) and a red membrane dye (DiI). The cells were then imaged live with a 30 sec time lapse on a DeltaVision microscope usnig a 20X oil objective. Upon fusion, the dyes transfer between the cells, and finally a triple-labeled syncytium forms. The membrane dye is observed to transfer first, which signifies hemifusion, The internal blue dye and GFP-tagged viral Gag then mix as the cytoplasm of each cell mixes with the other. Using a highly fusogenic Env truncated of its cytoplasmic tail, we can observe a 2-minute delay in pore opening upon CD9 overexpression. Experiments are under way to reproduce this using WT Env, where we expect to see a much stronger phenotype.
Animated GIF available at http://ge.tt/9f4UEfD1/v/0?c .
Lambelé M, Koppensteiner H, Symeonides M, Roy NH, Chan J, Schindler M, Thali M (2015) Vpu Is the Main Determinant for Tetraspanin Downregulation in HIV-1-Infected Cells. J Virol 89(6): 3247-55.
Roy NH, Lambelé M, Chan J, Symeonides M, Thali M (2014) Ezrin is a component of the HIV-1 virological presynapse and contributes to the inhibition of cell-cell fusion. J Virol 88(13): 7645-58.
Symeonides M, Lambelé M, Roy NH, Thali M (2014) Evidence showing that tetraspanins inhibit HIV-1-induced cell-cell fusion at a post-hemifusion stage. Viruses 6(3): 1078-90.
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