Biology subjects

Lai, K. K.

Publications and source records attributed to Lai, K. K..

7 recordsLinked to original sources

bioRxiv2025

IFITM1 and IFITM3 cooperate to restrict virus entry in endolysosomes

Interferon-induced transmembrane (IFITM) proteins are potent innate immune factors that restrict an array of viruses at the entry stage of infection. We previously characterized a GxxxG motif in the CD225 domain of human IFITM3 that mediates its multimerization, which is essential for the reduction of membrane fluidity by IFITM3 and for its antiviral activity against Influenza A virus. Here, using an unbiased approach coupling immunoprecipitation with mass spectrometry, we show that the GxxxG motif is also important for the interaction of IFITM3 with other proteins, including IFITM1. While IFITM1 is primarily regarded as a cell surface protein that restricts the entry of viruses fusing at the plasma membrane, this model is based mostly on overexpression studies and is at odds with some studies showing that it can restrict endocytic viruses. Here, we show that endogenous IFITM1 and IFITM3 co-reside in membranes of acidic late endosomes and lysosomes (endolysosomes) and form a protein-protein complex as determined by co-immunoprecipitation and proximity ligation assay. Knockdown of endogenous IFITM3 resulted in enhanced localization of IFITM1 at the plasma membrane, indicating that IFITM3 promotes IFITM1 localization to endolysosomes. To assess the antiviral protection conferred by endogenous IFITM1 and IFITM3 against viruses fusing at endolysosomal membranes, we measured cell entry mediated by the Influenza A fusogen hemagglutinin (HA). While knockdown of IFITM3 significantly boosted HA-mediated entry, combined knockdown of both IFITM3 and IFITM1 boosted entry even further. These results suggest that endogenous IFITM1 restricts Influenza A virus entry in a manner that is non-redundant with IFITM3, and that IFITM1 and IFITM3 inhibit virus entry in a cooperative manner.

microbiology↗

bioRxiv2025

Restriction of HIV-1 infectivity by interferon and IFITM3 is counteracted by Nef

The viral accessory protein Nef is a major determinant of HIV-1 pathogenicity in vivo. Nef is a multifunctional, immunomodulatory protein that downmodulates cell surface proteins, including CD4 and MHC class I (MHC-I) important for T-cell-mediated immunity. In addition, Nef also regulates cell-intrinsic immunity--Nef boosts the infectivity of virions produced and released from HIV-infected cells, at least in part, by counteracting the antiviral activity of transmembrane proteins SERINC3 and SERINC5. However, it has been reported that the enhancement of HIV-1 infectivity by Nef persists in certain cell lines deficient for SERINC3/5, revealing the existence of other Nef-sensitive host factors that impact HIV-1 infectivity. Here, we show that Nef proteins, especially those derived from many primary isolates of HIV-1, restore infectivity in interferon-treated cells and confer resistance to interferon-induced transmembrane protein 3 (IFITM3). IFITM3 is a restriction factor that reduces retroviral infectivity by incorporating into virions, inhibiting Envelope glycoprotein function, and reducing entry into cells. Using a primary isolate of Nef derived from HIV-1 clade C, we found that Nef interacts with IFITM3 in membranes and uses the endocytic adaptor protein AP-2 to counteract it. Furthermore, Nef reduced IFITM3 cell surface levels, increased IFITM3 levels in early endosomes, and reduced IFITM3 incorporation into HIV-1 virions. Nef also impaired IFITM3 oligomerization and restored membrane fluidity in IFITM3-expressing cells. The antiviral activity of IFITM3 and its inhibition by Nef were unaffected by SERINC5 knockdown, suggesting that the counteraction of IFITM3 represents a unique function of Nef. Our findings reveal a previously unrecognized immunomodulatory role for Nef in the setting of the interferon-induced antiviral state during HIV-1 infection.

microbiology↗

bioRxiv2025

Evolution of a fuzzy ribonucleoprotein complex in viral assembly

Previously we showed that the genetic diversity of SARS-CoV-2 nucleocapsid (N) protein explores a wide range of biophysical properties facilitated by non-local impact of point mutations to its intrinsically disordered regions (Nguyen et al., 2024). This includes modulation of self-association, such as the creation of a de novo binding interface through the P13L mutation characteristic of Omicron variants. In the present work we focus on the key function of N condensing viral RNA into ribonucleoprotein particles (RNPs) for viral assembly. Lacking high-resolution structural information, biochemical and biophysical approaches have revealed architectural principles of RNPs, which involve cooperative interactions of several protein-protein and protein-RNA interfaces, initiated through oligomerization of conserved transient helices in the central disordered linker of N. Here we study the impact of defining N-protein mutations in variants of concern on RNP formation, using biophysical tools, a virus-like particle assay, and reverse genetics experiments. We find convergent evolution in repeated, independent introduction of amino acid substitutions strengthening existing binding interfaces, compensating for other substitutions that promote viral replication but decrease RNP stability. Furthermore, we show that the P13L mutation of Omicron variants enhances RNP assembly and increases viral fitness. Overall, our data reveal RNP complexes to be highly variable not only in sequence and conformations, but also in thermodynamic and kinetic stability, with its pleomorphism affecting basic architectural principles. We hypothesize that the formation of polydisperse, fuzzy N-RNA clusters with multiple distributed weak binding interfaces optimizes reversible RNA condensation, while supporting host adaptation and allowing for a large sequence space to be explored.

biophysics↗

bioRxiv2024

Essential functions of Inositol hexakisphosphate (IP6) in Murine Leukemia Virus replication

We have investigated the function of inositol hexakisphosphate (IP6) and inositol pentakisphosphate (IP5) in the replication of murine leukemia virus (MLV). While IP6 is known to be critical for the life cycle of HIV-1, its significance in MLV remains unexplored. We find that IP6 is indeed important for MLV replication. It significantly enhances endogenous reverse transcription (ERT) in MLV. Additionally, a pelleting-based assay reveals that IP6 can stabilize MLV cores, thereby facilitating ERT. We find that IP5 and IP6 are packaged in MLV particles. However, unlike HIV-1, MLV depends upon the presence of IP6 and IP5 in target cells for successful infection. This IP6/5 requirement for infection is reflected in impaired reverse transcription observed in IP6/5-deficient cell lines. In summary, our findings demonstrate the importance of capsid stabilization by IP6/5 in the replication of diverse retroviruses; we suggest possible reasons for the differences from HIV-1 that we observed in MLV.

molecular biology↗

bioRxiv2023

Omicron Spike confers enhanced infectivity and interferon resistance to SARS-CoV-2 in human nasal tissue

Omicron emerged following COVID-19 vaccination campaigns, displaced previous SARS-CoV-2 variants of concern worldwide, and gave rise to lineages that continue to spread. Here, we show that Omicron exhibits increased infectivity in primary adult upper airway tissue relative to Delta. Using recombinant forms of SARS-CoV-2 and nasal epithelial cells cultured at the liquid-air interface, enhanced infectivity maps to the step of cellular entry and evolved recently through mutations unique to Omicron Spike. Unlike earlier variants of SARS-CoV-2, Omicron enters nasal cells independently of serine transmembrane proteases and instead relies upon metalloproteinases to catalyze membrane fusion. This entry pathway unlocked by Omicron Spike enables evasion of constitutive and interferon-induced antiviral factors that restrict SARS-CoV-2 entry following attachment. Therefore, the increased transmissibility exhibited by Omicron in humans may be attributed not only to its evasion of vaccine-elicited adaptive immunity, but also to its superior invasion of nasal epithelia and resistance to the cell-intrinsic barriers present therein.

microbiology↗

bioRxiv2022

CryoEM Structures of the Human HIV-1 Restriction Factor SERINC3 and Function as a Lipid Transporter

The host proteins SERINC3 and SERINC5 are HIV-1 restriction factors that reduce infectivity when incorporated into the viral envelope. The HIV-1 accessory protein Nef abrogates incorporation of SERINCs via binding to intracellular loop 4 (ICL4). CryoEM maps of full-length human SERINC3 and an ICL4 deletion construct reveal that hSERINC3 is comprised of two - helical bundles connected by a [~]40-residue, tilted, "crossmember" helix. The design resembles non-ATP-dependent lipid transporters. Consistently, purified hSERINCs reconstituted into proteoliposomes flip phosphatidylserine (PS), phosphatidylethanolamine and phosphatidylcholine. SERINC3 and SERINC5 reduce infectivity and expose PS on the surface of HIV-1 and also MLV, which is counteracted by Nef and GlycoGag, respectively. Antiviral activities by SERINCs and the scramblase TMEM16F correlate with the exposure of PS and with altered conformation of the envelope glycoprotein. We conclude that SERINCs are lipid transporters, and we demonstrate that lipid flipping is directly correlated with loss of infectivity. One Sentence SummaryThe HIV-1 restriction factor SERINC3 has a molecular design similar to non-ATP dependent lipid transporters, a function supported by the observation of flipping activity in proteoliposomes and exposure of phosphatidylserine on HIV-1 and MLV particles, which is correlated with loss of infectivity.

biophysics↗

bioRxiv2021

Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2

SARS-CoV-2 infection in immunocompromised individuals is associated with prolonged virus shedding and evolution of viral variants. Rapamycin and its analogs (rapalogs, including everolimus, temsirolimus, and ridaforolimus) are FDA-approved as mTOR inhibitors for the treatment of human diseases, including cancer and autoimmunity. Rapalog use is commonly associated with increased susceptibility to infection, which has been traditionally explained by impaired adaptive immunity. Here, we show that exposure to rapalogs increases susceptibility to SARS-CoV-2 infection in tissue culture and in immunologically naive rodents by antagonizing the cell-intrinsic immune response. By identifying one rapalog (ridaforolimus) that is less potent in this regard, we demonstrate that rapalogs promote Spike-mediated entry into cells by triggering the degradation of antiviral proteins IFITM2 and IFITM3 via an endolysosomal remodeling program called microautophagy. Rapalogs that increase virus entry inhibit the mTOR-mediated phosphorylation of the transcription factor TFEB, which facilitates its nuclear translocation and triggers microautophagy. In rodent models of infection, injection of rapamycin prior to and after virus exposure resulted in elevated SARS-CoV-2 replication and exacerbated viral disease, while ridaforolimus had milder effects. Overall, our findings indicate that preexisting use of certain rapalogs may elevate host susceptibility to SARS-CoV-2 infection and disease by activating lysosome-mediated suppression of intrinsic immunity. SignificanceRapamycin is an immunosuppressant used in humans to treat cancer, autoimmunity, and other disease states. Here, we show that rapamycin and related compounds promote the first step of the SARS-CoV-2 infection cycle--entry into cells--by disarming cell-intrinsic immune defenses. We outline the molecular basis for this effect by identifying a rapamycin derivative that is inactive, laying the foundation for improved mTOR inhibitors that do not suppress intrinsic immunity. We find that rapamycin analogs that promote SARS-CoV-2 entry are those that activate TFEB, a transcription factor that triggers the degradation of antiviral membrane proteins inside of cells. Finally, rapamycin administration to rodents prior to SARS-CoV-2 challenge results in enhanced viral disease, revealing that its use in humans may increase susceptibility to infection.

microbiology↗