Suchetana Mukhopadhyay

The assembly of enveloped viruses is a highly orchestrated process that depends on the coupling of multiple protein-protein interactions within a membrane environment. To gain mechanistic insight into this process, we use Chikungunya virus as a model system to study Alphavirus assembly, focusing on the interplay between core-spike and spike-spike interactions. We begin with coarse-grained molecular dynamics simulations to systematically explore how the symmetry of the nucleocapsid core, together with the relative strengths of spike-core and spike-spike interactions, influences budding efficiency and the emergence of icosahedral particle symmetry. Building on these computational results, we performed site-directed mutagenesis on Chikungunya virus 181/25 and examined the consequences for particle assembly and budding in cultured cells, as well as the impact of these mutations during in-cellulo assembly. Our results revealed that canonical core-spike interactions, while necessary, were not sufficient for successful assembly. Instead, lateral interactions among glycoproteins emerged as critical determinants of efficient budding, particle stability, and the maintenance of icosahedral symmetry. Together, these findings provided an integrated computational and experimental framework for understanding the molecular principles governing Alphavirus assembly.

Enveloped viruses are significant zoonotic disease threats with the potential to cause global pandemics. We identified a class of small-molecule sulfur-containing antiviral compounds (XM series) that broadly inhibit enveloped viruses. Multidisciplinary approaches revealed that XM compounds alter the viral membrane lipid chemical composition, enhance membrane order within the hydrophobic bilayer, and increase membrane phase transition temperatures. This mechanism inhibits membrane fusion and viral entry, while leaving the viral glycoproteins and genomes largely unaffected. Leveraging these unique properties, we develop a proof-of-concept whole inactivated influenza virus (IIV) vaccine using XM-01 (XM-01-IIV). In a mouse model, XM-01-IIV elicit significantly enhanced neutralizing antibody responses against hemagglutinin and neuraminidase compared to traditional paraformaldehyde-inactivated vaccines. Further, XM-01-IIV reduces morbidity and mortality following influenza challenge, achieving protection comparable to live virus vaccination. This promising class of broadly acting antivirals can be highly impactful in the development of highly potent inactivated vaccines for enveloped viruses.

Curriculum guidelines for virology are needed to best guide student learning due to the continuous and ever-increasing volume of virology information, the need to ensure that undergraduate and graduate students have a foundational understanding of key virology concepts, and the importance in being able to communicate that understanding to both other virologists and nonvirologists. Such guidelines, developed by virology educators and the American Society for Virology Education and Career Development Committee, are described herein.

The constrained nature of viral genomes has allowed a translational sleight of hand known as −1 Programmed Ribosomal Frameshifting (−1 PRF) to flourish. Numerous studies have sought to tease apart the mechanisms and implications of −1PRF utilizing a few techniques.

Alphaviruses cause significant outbreaks of febrile illness and debilitating multi-joint arthritis for prolonged periods after initial infection. We have previously reported that several host hnRNP proteins bind to the Sindbis virus (SINV) RNAs, and disrupting the sites of these RNA–protein interactions results in decreased viral titers in tissue culture models of infection. Intriguingly, the primary molecular defect associated with the disruption of the hnRNP interactions is enhanced viral structural protein expression; however, the precise underlying mechanisms spurring the enhanced gene expression remain unknown. Moreover, our previous efforts were unable to functionally dissect whether the observed phenotypes were due to the loss of hnRNP binding or the incorporation of polymorphisms into the primary nucleotide sequence of SINV. To determine if the loss of hnRNP binding was the primary cause of attenuation or if the disruption of the RNA sequence itself was responsible for the observed phenotypes, we utilized an innovative protein tethering approach to restore the binding of the hnRNP proteins in the absence of the native interaction site. Specifically, we reconstituted the hnRNP I interaction by incorporating the 20nt bovine immunodeficiency virus transactivation RNA response (BIV-TAR) at the site of the native hnRNP I interaction sequence, which will bind with high specificity to proteins tagged with a TAT peptide. The reestablishment of the hnRNP I–vRNA interaction via the BIV-TAR/TAT tethering approach restored the phenotype back to wild-type levels. This included an apparent decrease in structural protein expression in the absence of the native primary nucleotide sequences corresponding to the hnRNP I interaction site. Collectively, the characterization of the hnRNP I interaction site elucidated the role of hnRNPs during viral infection.

Alphaviruses, which can cause disease when spread to humans by mosquitoes, have been classified as emerging pathogens, with infections occurring worldwide. The spikes on the surface of the alphavirus particle are absolutely required for the virus to enter a new host cell and initiate an infection.

Since its introduction into the Caribbean in October 2013, CHIKV has rapidly spread to almost the entire neotropical region. However, its potential to further spread globally, including into more temperate climates, depends in part on its ability to be transmitted efficiently by Aedes albopictus , which can survive colder winters than A. aegypti .

Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism that enables the synthesis of multiple polypeptides from a single transcript. During translation of the alphavirus structural polyprotein, the efficiency of −1PRF is coordinated by a ‘slippery’ sequence in the transcript, an adjacent RNA stem–loop, and a conformational transition in the nascent polypeptide chain. To characterize each of these effectors, we measured the effects of 4530 mutations on −1PRF by deep mutational scanning. While most mutations within the slip-site and stem–loop reduce the efficiency of −1PRF, the effects of mutations upstream of the slip-site are far more variable. We identify several regions where modifications of the amino acid sequence of the nascent polypeptide impact the efficiency of −1PRF. Molecular dynamics simulations of polyprotein biogenesis suggest the effects of these mutations primarily arise from their impacts on the mechanical forces that are generated by the translocon-mediated cotranslational folding of the nascent polypeptide chain. Finally, we provide evidence suggesting that the coupling between cotranslational folding and −1PRF depends on the translation kinetics upstream of the slip-site. These findings demonstrate how −1PRF is coordinated by features within both the transcript and nascent chain.

Structural viral proteins have multiple roles during entry and assembly. The capsid protein (CP) of alphaviruses has one domain that interacts with the viral genome and another domain that interacts with the E2 spike protein.