We study the mechanisms of entry and fusion of enveloped viruses, such as HIV-1, Influenza, Lassa, Ebola and other human pathogenic viruses. The use of functional assays, including real-time visualization of single virus fusion, enables monitoring the entry process of a minority of relevant particles undergoing fusion and establishing infection. We develop and apply novel approaches to fluorescently labeling different viral components and visualize their release into the cytoplasm, redistribution within the cell membranes and transport to the nucleus. Through the use of these techniques, along with long-term live cell imaging, we visualize the entire virus entry process that culminates in infection. We also work on implementing correlative light-electron microscopy technique to gain structural information regarding the key intermediate steps of virus entry. Our group is interested in delineating the mechanisms of antiviral activity of host restriction factors that target virus entry/fusion and identifying novel small molecule inhibitors of viral fusion.
Our laboratory has characterized the mechanism of HIV-1 Env glycoprotein-mediated membrane fusion. Through the usage of various interventions and fusion inhibitors, we identified key intermediate steps of this process, such as a target membrane-inserted pre-hairpin intermediate, and showed that the requisite number of gp41 completes refolding into the final 6-helix bundle structure after the formation of a fusion pore (Figure 1).
Single particle tracking revealed that HIV-1, which has long been thought to infect host cells by fusing directly with the plasma membrane, enters several target cells via endocytosis and pH-independent fusion with endosomes (Figure 2 and Movie 1). In contrast, HIV-1 fusion initiated at the cell surface did not progress beyond a lipid mixing stage. These surprising findings implicated yet unknown endocytic trafficking factors in facilitating HIV fusion. Through a targeted shRNA screen, we have since identified several membrane trafficking proteins as essential factors for HIV-1 fusion.
In spite of the apparent lack of HIV-1 fusion at the plasma membrane, viruses “wedged” between two adjacent cells can mediate cell-cell fusion. Unlike HIV-cell fusion, this HIV-mediated cell-cell fusion (referred to as “fusion-from-without”, FFWO) is very inefficient and is highly dependent on actin dynamics. Actin-dependence suggests a role for an external cell-generated force in HIV-1 fusion at the cell surface. We thus hypothesize that HIV-1 relies on mechanical tension in a cell membrane to dilate nascent fusion pores and release its genome into the cytoplasm (Figure 3). This hypothesis is currently being tested in collaboration with Drs. Khalid Salaita (Emory) and Cheng Zhu (Georgia Tech).
In the absence of HIV/SIV Nef expression, SERINC5 incorporates into budding virions and inhibits viral fusion, but the mechanism of restriction remains unclear. Envelope glycoproteins (Env) from different HIV-1 isolates exhibit a broad range of sensitivity to SERINC5. We have recently shown that incorporation of SERINC5 into virions inhibits the formation of small fusion pores between viruses and cells. We found that SERINC5 incorporation enhances the exposure of conserved HIV-1 gp41 domains, sensitizes the virus to neutralizing antibodies and promotes spontaneous inactivation of Env (Figure 1). Current efforts are focused on delineating the mechanism by which SERINC5 exerts these effects, including analysis of intraviral Env distribution in SERINC5 vs control virions by super-resolution imaging (Figure 2).
Disassembly of the cone-shaped HIV-1 capsid (uncoating) after virus-cell fusion is a prerequisite for establishing infection. We have developed a novel strategy to visualize HIV uncoating that is based on a fluorescently tagged oligomeric form of a capsid-binding host protein cyclophilin A (CypA-DsRed). CypA-DsRed, which is specifically packaged into virions through the high-avidity binding to the HIV-1 CA, does not compromise the infectivity. Through the use of this novel CA marker and long-term live cell imaging we tracked single virus cores and showed that productive uncoating that leads to infection occurs at the nuclear pore (Figure 1 and Movies 1 and 2). We demonstrated the role of CA in protecting the viral complexes from degradation in the cytoplasm and for docking at the nuclear pore. We also found a surprisingly strong correlation between subsequent disappearance of fluorescent integrase complexes in the nucleus and virus integration.
Interferon-induced transmembrane proteins (IFITMs, Figure 1) inhibit infection of diverse enveloped viruses, including the influenza A virus (IAV), West Nile, Ebola and other viruses. We have examined the mechanism of IAV restriction by IFITM3 protein using direct virus-cell fusion assay and single virus imaging in live cells. IFITM3 does not inhibit lipid mixing, but abrogates the release of viral content into the cytoplasm (Figure 1). IFITM3’s ability to block fusion pore formation at a post-hemifusion stage suggests that this protein stabilizes the cytoplasmic leaflet of endosomal membranes without adversely affecting the lumenal leaflet. Alternatively, IFITM3 may redirect IAV to a non-productive pathway by promoting fusion with intralumenal vesicles within multivesicular bodies/late endosomes.
In collaboration with Dr. Elizabeth Wright, we combined live cell single virus fluorescence microscopy with cryo-electron tomography of the viral fusion sites. Proof-of-concept correlated images of single HIV-1 particles located in a cell protrusion are shown in Figure 1.
Mariana Marin (Assistant Professor) Ashwanth Francis (Instructor)
Christine Lee (Postdoc) Yen-Cheng Chen (Postdoc)
Junghwa Choi (graduate student) Kristina Rowland (Res. Specialist)
Matthew Prellberg (Res. Specialist)
Caleb Mason, Chetan Sood, Krishna Suddala, Charline Giroud, Tanay Desai, Jennifer Spence, Yoon-Hyeun Oum, Lusine Demirkhanyan, Naoyuki Kondo, Rolf Suter, Kiran Verma, Michelle de la Vega, Sergi Padilla-Parra, Surbhi Jain, Nishi Sharma, Kosuke Miyauchi, Olga Latinovic, Yuri Kim, Grant Jones, Gennadiy Novitskiy, Naveen Jha, Patrick Millet, Victoria Saakian, Vladimir Morozov, Sergey Cheresiz, Michael Leung, Levon Abrahamyan
Nivriti Galhaut, David Gevorgyan, Pedro Matos