We study the entry mechanisms of enveloped viruses, such as HIV, influenza virus and Avian Sarcoma and Leukosis Virus (ASLV), that initiate infection by merging their membrane with the target cell membrane. The use of functional assays, including real-time visualization of single virus fusion, circumvents the major obstacle for these studies - the fact that only a small fraction of viruses internalized by cells undergoes fusion and establishes infection. We label different viral components with fluorescent probes and visualize their release into the cytoplasm, redistribution within the cell membranes and transport to the nucleus. These labeling and single virus imaging approaches enable detection of viral hemifusion, formation of a small fusion pore and release of the nucleocapsid (pore enlargement). There is increasing appreciation that host factors other than receptors are essential for establishing infection, while other factors block viral fusion. We are interested in delineating the mechanisms of antiviral activity of host restriction factors that target virus entry and identifying novel small molecule inhibitors of viral fusion.

Our laboratory has identified intermediate stages of fusion mediated by the influenza hemagglutinin, HIV Env glycoprotein and ASLV Env. Current efforts are focused on delineating the HIV-1 entry pathways into cells. Single particle tracking revealed that HIV, 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. Through a targeted shRNA screen, we have since identified several endocytic trafficking proteins as essential factors for HIV fusion and are investigating their roles in the entry process. In spite of the apparent lack of HIV fusion at the plasma membrane, viruses “wedged” between two adjacent cells can mediate cell-cell fusion. Unlike HIV-cell fusion, HIV-mediated cell-cell fusion (referred to as fusion-from-without, FFWO) is very inefficient and highly dependent on actin dynamics. Actin-dependence suggests a role for an external cell-generated force in HIV-1 fusion at the cell surface. Particles adhered to the plasma membranes of both neighboring cells – a condition that is satisfied in FFWO, but not for viruses bound to a single cell – could be subjected to forces that likely originate from Env-mediated signaling and actin remodeling. We thus hypothesize that HIV relies on mechanical tension in a cell membrane to dilate nascent fusion pores and release its genome into the cytoplasm. This hypothesis is currently being tested in collaboration with Dr. Khalid Salaita (Emory) and Cheng Zhu (Georgia Tech).

In collaboration with the Emory Chemical Biology Discovery Center (Drs. Yuhong Du and Haian Fu), we have carried out high-throughput screening for small molecule HIV fusion inhibitors. The hits from a 100,000 compound library are being analyzed and validated. Among the hits were several purinergic receptor antagonists as inhibitors of HIV fusion. We have shown that NF279, a P2X1 receptor antagonist, blocks the binding of both CCR5 and CXCR4 coreceptors by the Env-CD4 complexes. 

In the absence of HIV/SIV Nef expression, SERINC5 incorporates into budding virions and inhibits viral fusion. Envelope glycoproteins (Env) from different HIV-1 isolates exhibit a broad range of sensitivity to SERINC5, and the mechanism of restriction remains unclear. 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 promotes spontaneous functional inactivation of sensitive but not resistant Env glycoproteins, and that SERINC5 incorporation enhances the exposure of the conserved gp41 domains and sensitizes the virus to neutralizing antibodies and gp41-derived inhibitory peptides. These results imply that SERINC5 blocks HIV-1 infection at a step prior to small fusion pore formation by selectively inactivating Env and delaying conformational transitions of the HIV-1 glycoprotein en route to fusion.

 

Disassembly of the cone-shaped HIV 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 is specifically packaged into virions through the high-avidity binding to the HIV-1 capsid, does not compromise the infectivity and remains associated till uncoating. We found that CypA-DsRed loss from individual post-fusion cores is accelerated by reverse transcription and is modulated by the capsid (CA) mutations that alter the core stability. The CypA-DsRed based imaging assay showed biphasic HIV-1 uncoating, with a large number of cores shedding the CA marker shortly after fusion and a small fraction of cores undergoing gradual uncoating at late times post-infection. We are currently delineating CypA-DsRed interactions with the HIV core, using imaging and structural approaches, and assessing the effect of CA-interacting host factors on the rate of uncoating. 

Interferon-induced transmembrane proteins (IFITMs) 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 did not inhibit lipid mixing, but abrogated the release of viral content into the cytoplasm. 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 fusion to a non-productive pathway by promoting fusion with intralumenal vesicles within multivesicular bodies/late endosomes. We are currently delineating the mechanisms of inhibition of viral fusion, using IFITM-sensitive (IAV) and IFITM-resistant (arenaviruses) viruses.

http://www.ncbi.nlm.nih.gov/sites/myncbi/gregory.melikian.1/bibliography/40733965/public/?sort=date&direction=descending

Mariana Marin (Assistant Professor)  Chetan  Sood (Posdtoc)  Ashwanth Francis (Postdoc)

  Krishna Suddala  (Postdoc)  Caleb Mason (Research Specialist)  Junghwa Choi (graduate student)

Kia Thomas, Mialovena Exume, Kenyanna Taylor, Margaret Black.

Charline Giroud, Tanay Desai, Jennifer Spence, Yoon-Hyeun Oum, Lusine Demirkhanyan, Naoyuki Kondo, 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