Biology subjects

Melikyan, G. B.

Publications and source records attributed to Melikyan, G. B..

7 recordsLinked to original sources

bioRxiv2025

Structural, biophysical, and virological mechanistic characterization of HIV-1 capsid-targeting antivirals

Due to its significant role in virus replication, the HIV capsid is an attractive antiviral target. This is validated by the recent clinical approval of lenacapavir for both treatment and pre-exposure prophylaxis (PrEP). PF74 is a well-characterized capsid-targeting antiviral that was discontinued in further study due to potency and metabolic issues. We hypothesized that making chemical modifications at certain sites of PF74 could result in capsid-targeting antivirals with improved potency and bioavailability. Our cumulative studies show that making changes at the R1 and R3 positions of PF74 results in compounds with increased antiviral potency, increased stability of wild-type HIV capsid hexamers and virions, tighter binding to wild-type HIV capsid hexamer compared to PF74, and different interactions at the "FG" binding site of capsid compared to PF74. These data provide insights into the design of future capsid-targeting antivirals relevant for clinical use.

biochemistry↗

bioRxiv2025

SERINC5 co-expressed with HIV-1 Env or present in a target membrane destabilizes small fusion pores leading to their collapse

SERINC5 is a multipass membrane protein that reduces infectivity of retroviruses and other enveloped viruses through incorporation into budding virions and inhibiting viral fusion. Several modes of anti-HIV activity of SERINC5 have been reported, including binding to Env glycoprotein, induction of conformational changes and destabilization of Env, as well as disruption of trans-membrane asymmetry of viral envelope. All these reported anti-HIV mechanisms involve SERINC5 incorporation into progeny virions, while there is very little information regarding a potential antiviral activity of SERINC5 in target cells. Here, we show that SERINC5 expressed in target cells efficiently inhibits fusion with cells expressing sensitive but not resistant HIV-1 strains. We show that this activity does not result from downregulation of CD4 or coreceptors on target cells or interference with the formation of ternary Env/CD4/coreceptor complexes. We further demonstrate that SERINC5 destabilizes small fusion pores causing their collapse and that this block can be rescued by incorporation of phosphatidylserine to either effector (Env-expressing) or target cells. Interestingly, we did not detect a significant reduction of HIV-1 pseudovirus fusion with SERINC5 expressing target cells, while virus-mediated cell-cell fusion (fusion-from-without) was inhibited, suggesting a potential role of virus entry pathway in SERINC5 restriction. Collectively, our results reveal a novel mechanism of inhibition of HIV-1 fusion by SERINC5 through destabilization of small fusion pores in a manner that depends on lipid composition. ImportanceSERINC5 incorporates into virions produced by infected cells and inhibits viral fusion with target cells through a poorly understood mechanism. Here, we show that SERINC5 blocks HIV-1 Env mediated cell fusion when expressed in either effector (Env-expressing) or target cells. Inhibition of Env-mediated fusion by SERINC5 expressed in target cells is not through reduction in receptor or coreceptor expression or interference with Envs ability to engage a requisite number of receptors and coreceptors. We demonstrate that the block of fusion is at a post-hemifusion stage of small fusion pores that collapse when SERINC5 is present in effector or target membrane and that this block is rescued by incorporation of specific anionic lipids, such as phosphatidylserine. These findings reveal a previously unappreciated mode of HIV-1 restriction through destabilization of small fusion pores that occurs irrespective of SERINC5 localization in either of fusing membranes.

microbiology↗

bioRxiv2025

Attenuation of IFITM proteins antiviral activity through sequestration into intraluminal vesicles of late endosomes

Interferon-induced transmembrane proteins (IFITMs) inhibit the entry of diverse enveloped viruses. The spectrum of antiviral activity of IFITMs is largely determined by their subcellular localization. IFITM1 localizes to and primarily blocks viral fusion at the plasma membrane, while IFITM3 prevents viral fusion in late endosomes by accumulating in these compartments. We have previously reported that cyclosporine A (CsA) treatment relieves the fusion block for the Influenza A virus, likely by relocating IFITM1 and IFITM3 from the plasma membrane and endosomes, respectively, to the Golgi area. Here, we report the existence of at least two distinct pools of IFITMs in CsA treated cells. While immunostaining of CsA treated cells using mild permeabilization agents, such as digitonin, suggests preferential IFITM localization at the Golgi apparatus, a harsher permeabilization protocol reveals a large, previously unidentified pool of IFITMs in late endosomes. Notably, IFITM redistribution was not associated with its degradation. A disproportionate loss of antibody access to the cytoplasmic N-terminus compared to the extracellular C-terminus of IFITMs after CsA treatment is consistent with sequestration of the N-terminal domain inside intraluminal vesicles of late endosomes. Accordingly, super-resolution microscopy reveals that CsA induces IFITM3 redistribution from the periphery to the interior of late endosomes. Together, our results imply that IFITMs relocate to intraluminal vesicles of late endosomes in the presence of CsA, thereby enabling viral fusion with the limiting membrane of these compartments. Our findings highlight the critical role of IFITM trafficking in antiviral defense and suggest a novel mechanism through which CsA modulates the cells susceptibility to viral infections.

microbiology↗

bioRxiv2023

Rolosense: Mechanical detection of SARS-CoV-2 using a DNA-based motor

Assays detecting viral infections play a significant role in limiting the spread of diseases such as SARS-CoV-2. Here we present Rolosense, a virus sensing platform that transduces the motion of synthetic DNA-based motors transporting 5-micron particles on RNA fuel chips. Motors and chips are modified with virus-binding aptamers that lead to stalling of motion. Therefore, motors perform a "mechanical test" of viral target and stall in the presence of whole virions which represents a unique mechanism of transduction distinct from conventional assays. Rolosense can detect SARS-CoV-2 spiked in artificial saliva and exhaled breath condensate with a sensitivity of 103 copies/mL and discriminates among other respiratory viruses. The assay is modular and amenable to multiplexing, as we demonstrated one-pot detection of influenza A and SARS-CoV-2. As a proof-of-concept, we show readout can be achieved using a smartphone camera in as little as 15 mins without any sample preparation steps. Taken together, mechanical detection using Rolosense can be broadly applied to any viral target and has the potential to enable rapid, low-cost, point-of-care screening of circulating viruses.

biophysics↗

bioRxiv2023

Fusogenic Structural Changes in Arenavirus Glycoproteins are Associated with Viroporin Activity

Many enveloped viruses enter host cells by fusing with acidic endosomes. The fusion activity of multiple viral envelope glycoproteins does not generally affect viral membrane permeability. However, fusion induced by the Lassa virus (LASV) glycoprotein complex (GPc) is always preceded by an increase in viral membrane permeability and the ensuing acidification of the virion interior. Here, systematic investigation of "leaky" LASV fusion using single pseudoparticle tracking in live cells reveals that the change in membrane barrier function is associated with the fusogenic conformational reorganization of GPc. We show that a small- molecule fusion inhibitor or mutations that impair viral fusion by interfering with GPc refolding into the post-fusion structure prevent the increase in membrane permeability. We find that the increase in virion membrane permeability occurs early during endosomal maturation and is facilitated by virus-cell contact. This increase is observed using diverse arenavirus glycoproteins, whether presented on lentivirus-based pseudoviruses or arenavirus-like particles, and in multiple different cell types. Collectively, these results suggest that conformational changes in GPc triggered by low pH and cell factor binding are responsible for virion membrane permeabilization and acidification of the virion core prior to fusion. We propose that this viroporin-like activity may augment viral fusion and/or post-fusion steps of infection, including ribonucleoprotein release into the cytoplasm. Authors summaryFusion of enveloped virus with host cell membranes is mediated by extensive conformational changes in viral glycoproteins, triggered by binding to cognate receptors and/or by exposure to acidic pH in the maturing endosome. We have previously reported that, unlike many other viral glycoproteins, pH-triggered endosomal fusion by the Lassa virus viral glycoprotein complex (GPc) is preceded by a mild permeabilization of the viral membrane. Here, we provide evidence that this activity is associated with early fusogenic changes in the GPc and is markedly enhanced by virus-cell contact. Permeabilization is induced by the glycoproteins of diverse arenaviruses and occurs in multiple target cell types. Based on these observations, we propose that, by analogy to the influenza virus M2 channel, membrane permeabilization and the resultant acidification of the arenavirus interior facilitate viral fusion and/or post-fusion steps of infection.

microbiology↗

bioRxiv2022

Human LAMP1 accelerates Lassa virus fusion and potently promotes fusion pore dilation upon forcing viral fusion with non-endosomal membrane

Lassa virus (LASV) cell entry is mediated by the interaction of the virus glycoprotein complex (GPC) with alpha-dystroglycan at the cell surface followed by binding to LAMP1 in late endosomes. However, LAMP1 is not absolutely required for LASV fusion, as this virus can infect LAMP1-deficient cells. Here, we used LASV GPC pseudoviruses, LASV virus-like particles and recombinant lymphocytic choriomeningitis virus expressing LASV GPC to investigate the role of human LAMP1 (hLAMP1) in LASV fusion with human and avian cells expressing a LAMP1 ortholog that does not support LASV entry. We employed a combination of single virus imaging and virus population-based fusion and infectivity assays to dissect the hLAMP1 requirement for initiation and completion of LASV fusion that culminates in the release of viral ribonucleoprotein into the cytoplasm. Unexpectedly, ectopic expression of hLAMP1 accelerated the kinetics of small fusion pore formation, but only modestly increased productive LASV fusion and infection of human and avian cells. To assess the effects of hLAMP1 in the absence of requisite endosomal host factors, we forced LASV fusion with the plasma membrane by applying low pH. Unlike the conventional LASV entry pathway, ectopic hLAMP1 expression dramatically promoted the initial and full dilation of pores formed through forced fusion at the plasma membrane. We further show that, while the soluble hLAMP1 ectodomain accelerates the kinetics of nascent pore formation, it fails to promote efficient pore dilation, suggesting the hLAMP1 transmembrane domain is involved in this late stage of LASV fusion. These findings reveal a previously unappreciated role of hLAMP1 in promoting dilation of LASV fusion pores, which is difficult to ascertain for endosomal fusion where several co-factors, such as bis(monoacylglycero)phosphate, likely regulate LASV entry. Author SummaryLassa virus (LASV) enters cells via fusion with acidic endosomes mediated by the viral glycoprotein complex (GPC) interaction with the intracellular receptor LAMP1. However, the requirement for LAMP1 is not absolute, as LASV can infect avian cells expressing a LAMP1 ortholog that does not interact with GPC. To delineate the role of LAMP1 in LASV entry, we developed assays to monitor the formation of nascent fusion pores, as well as their initial and complete dilation to sizes that allow productive infection of avian cells by LASV GPC pseudoviruses. This novel approach provided unprecedented details regarding the dynamics of LASV fusion pores and revealed that ectopic expression of human LAMP1 in avian cells leads to a marked acceleration of fusion but modestly increases the likelihood of complete pore dilation and infection. In contrast, human LAMP1 expression dramatically enhanced the propensity of nascent pores to fully enlarge when LASV fusion with the plasma membrane was forced by exposure to low pH. We conclude that, whereas the role of LAMP1 in LASV fusion is confounded by an interplay between multiple endosomal factors, the plasma membrane is a suitable target for mechanistic dissection of the roles of endosomal factors in LASV entry.

microbiology↗

bioRxiv2020

Interferon-Induced Transmembrane Protein 3 Blocks Fusion of Diverse Enveloped Viruses by Locally Altering Mechanical Properties of Cell Membranes

Interferon-induced transmembrane protein 3 (IFITM3) potently inhibits entry of diverse enveloped viruses by trapping the viral fusion at a hemifusion stage, but the underlying mechanism remains unclear. Here, we show that recombinant IFITM3 reconstituted into lipid vesicles induces negative membrane curvature and that this effect maps to its small amphipathic helix (AH). We demonstrate that AH: (i) partitions into lipid-disordered domains where IAV fusion occurs, (ii) induces negative membrane curvature, and (iii) increases lipid order and membrane stiffness. Additionally, replacing the IFITM3 AH with AH from an unrelated protein did not compromise its antiviral activity. These effects on membrane properties correlate with the fusion-inhibitory activity, as exogenous addition of AH to insulin-producing cells reduces glucose-stimulated insulin secretion. Our results thus imply that IFITM3 inhibits the transition from hemifusion to full fusion by imposing an unfavorable membrane curvature and increasing the order and stiffness of the cytoplasmic leaflet of endosomal membranes.

biophysics↗