Biology subjects

Shmulevitz, M.

Publications and source records attributed to Shmulevitz, M..

6 recordsLinked to original sources

bioRxiv2024

Mathematical Modelling of Reoviruses in Cancer Cell Cultures

Oncolytic virotherapy has emerged as a potential cancer therapy, utilizing viruses to selectively target and replicate within cancer cells while preserving normal cells. In this paper, we investigate the oncolytic potential of unmodified reovirus T3wt relative to a mutated variant SV5. In animal cancer cell monolayer experiments it was found that SV5 was more oncolytic relative to T3wt. SV5 forms larger sized plaques on cancer cell monolayers and spreads to farther distances from the initial site of infection as compared to T3wt. Paradoxically, SV5 attaches to cancer cells less efficiently than T3wt, which lead us to hypothesize that there might be an optimal binding affinity with maximal oncolytic activity. To understand the relationship between the binding process and virus spread for T3wt and SV5, we employ mathematical modelling. A reaction-diffusion model is applied, which is fit to the available data and then validated on data that were not used for the fit. Analysis of our model shows that there is an optimal binding rate that leads to maximum viral infection of the cancer monolayer, and we estimate this value for T3wt and SV5. Moreover, we find that the viral burst size is an important parameter for viral spread, and that a combination of efficient binding and large burst sizes is a promising direction to further develop anti-cancer viruses.

cancer biology↗

bioRxiv2022

Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly.

Reoviridae virus family members, such as mammalian orthoreovirus (reovirus), encounter a unique challenge during replication. To hide the dsRNA from host recognition, the genome remains encapsidated in transcriptionally active proteinaceous core capsids that transcribe and release +RNA. De novo +RNAs and core proteins must repeatedly assemble into new progeny cores in order to logarithmically amplify replication. Reoviruses also produce outercapsid (OC) proteins {micro}1, {sigma}3 and {sigma}1 that assemble onto cores to create highly stable infectious full virions. Current models of reovirus replication position amplification of transcriptionally-active cores and assembly of infectious virions in shared factories, but we hypothesized that since assembly of OC proteins would halt core amplification, OC assembly is somehow regulated. Using kinetic analysis of virus +RNA, core and OC proteins, core assembly and whole virus assembly, assembly of OC proteins was found to be temporally delayed. All viral RNAs and proteins were made simultaneously, eliminating the possibility that delayed OC RNAs or proteins account for delayed OC assembly. High resolution fluorescence and electron microscopy revealed that core amplification occurred early during infection at peripheral core-only factories, while all OC proteins associated with lipid droplets (LDs) that coalesced near the nucleus in a {micro}1-dependent manner. Core-only factories transitioned towards the nucleus despite cycloheximide-mediated halting of new protein expression, while new core-only factories developed in the periphery. As infection progressed, OC assembly occurred at LD-and nuclear-proximal factories. Silencing of OC {micro}1 expression with siRNAs led to large factories that remained further from the nucleus, implicating {micro}1 in the transition to perinuclear factories. Moreover, late during infection, +RNA pools largely contributed to the production of de-novo viral proteins and fully-assembled infectious viruses. Altogether the results suggest an advanced model of reovirus replication with spatiotemporal segregation of core amplification, OC complexes and fully assembled virions. NON-TECHNICAL AUTHOR SUMMARYIt is important to understand how viruses replicate and assemble to discover antiviral therapies and to modify viruses for applications like gene therapy or cancer therapy. Reovirus is a harmless virus being tested as a cancer therapy. Reovirus has two coats of proteins, an inner coat and an outer coat. To replicate, reovirus particles need only the inner coat, but to become infectious they require the outer coat. Strangely, inner and outer coat proteins are all made by the virus at once, so it was unknown what determines whether newly made viruses will contain just the inner coat to continue to replicate, or both coats to transmit to new hosts. Our experiments reveal that the inner coat proteins are located in a different area of an infected cell versus the outer coat proteins. The location therefore determines if the newly made viruses contain just the inner coat versus both coats. Reoviruses have evolved extravagant mechanisms to be able to efficiently take on the best composition required for replication and transmission.

microbiology↗

bioRxiv2022

p38 MAPK Signaling Enhances Reovirus Replication by Facilitating Efficient Virus Entry, Virus Capsid Uncoating and Post-Uncoating Steps.

Mammalian orthoreovirus serotype 3 Dearing an orphan human virus currently being pursued as an oncolytic virus in multiple phase I/II clinical trials. Previous clinical trials have emphasized the importance of patient pre-screening for prognostic markers to improve therapeutic success. However, currently, only generic cancer markers such as EGFR, Hras, Kras, Nras, Braf and/or p53 are utilized and have exhibited limited benefit in predicting therapeutic efficacy. Utilization of more specific markers that influence specific steps during reovirus replication could prove beneficial as prognostic markers. This study delineated the role of p38 MAPK signaling during reovirus infection and illustrated a connection between specific p38 MAPK isoforms and reovirus infection. Using a panel of specific p38 MAPK inhibitors and an inactive inhibitor analogue, we demonstrated that p38 MAPK signaling is essential for establishment of reovirus infection by enhancing reovirus endocytosis, facilitating efficient reovirus uncoating at the endo-lysosomal stage, and augmenting post uncoating replication steps. Using a broad panel of human breast cancer cell lines, we observed susceptibility of reovirus infection corresponded with virus binding and uncoating efficiency, which was strongly correlated with status of the p38{beta} isoform. Together, our study proposes p38{beta} as a potential prognostic marker for early stages of reovirus infection that are crucial to establishment of successful reovirus infection.

cell biology↗

bioRxiv2019

Breast tumor-associated metalloproteases restrict reovirus oncolysis by cleaving the σ1 cell-attachment protein, and can be overcome by mutation of σ1

Reovirus is undergoing clinical testing as an oncolytic therapy for breast cancer. Given that reovirus naturally evolved to thrive in enteric environments, we sought to better understand how breast tumor microenvironments impinge on reovirus infection. Reovirus was treated with extracellular extracts generated from polyoma virus middle T-antigen-derived mouse breast tumors. Unexpectedly, these breast tumor extracellular extracts inactivated reovirus, reducing infectivity of reovirus particles by 100-fold. Mechanistically, inactivation was attributed to proteolytic cleavage of the viral cell attachment protein {sigma}1, which diminished virus binding to sialic acid-low tumor cells. Among various specific protease class inhibitors and metal ions, EDTA and ZnCl2 effectively modulated {sigma}1 cleavage, indicating that breast tumor-associated zinc-dependent metalloproteases are responsible for reovirus inactivation. Moreover, media from MCF7, MB468, MD-MB-231 and HS578T breast cancer cell lines recapitulated {sigma}1 cleavage and reovirus inactivation, suggesting that inactivation of reovirus is shared among mouse and human breast cancers, and that breast cancer cells in by themselves can be a source of reovirus-inactivating proteases. Binding assays and quantification of sialic acid (SA) levels on a panel of cancer cells showed that truncated {sigma}1 reduced virus binding to cells with low surface SA. To overcome this restriction, we generated a reovirus mutant with a mutation (T249I) in {sigma}1 that prevents {sigma}1 cleavage and inactivation by breast tumor-associated proteases. The mutant reovirus showed similar replication kinetics in tumorigenic cells, equivalent toxicity as wild-type reovirus in a severely compromised mouse model, and increased tumor titers. Overall, the data shows that tumor microenvironments have the potential to reduce infectivity of reovirus.\n\nSIGNIFICANCEWe demonstrate that metalloproteases in breast tumor microenvironments can inactivate reovirus. Our findings expose that tumor microenvironment proteases could have negative impact on proteinaceous cancer therapies such as reovirus, and that modification of such therapies to circumvent inactivation by tumor metalloproteases merits consideration.

microbiology↗

bioRxiv2019

Generation of Recombinant Rotavirus Expressing NSP3-UnaG Fusion Protein by a Simplified Reverse Genetics System

Rotavirus is a segmented double-stranded (ds)RNA virus that causes severe gastroenteritis in young children. We have established an efficient simplified rotavirus reverse genetics (RG) system that uses eleven T7 plasmids, each expressing a unique simian SA11 (+)RNA, and a CMV support plasmid for the African swine fever virus NP868R capping enzyme. With the NP868R-based system, we generated recombinant rotavirus (rSA11/NSP3-FL-UnaG) with a genetically modified 1.5-kB segment 7 dsRNA that encodes full-length NSP3 fused to UnaG, a 139-aa green fluorescent protein (FP). Analysis of rSA11/NSP3-FL-UnaG showed that the virus replicated efficiently and was genetically stable over 10 rounds of serial passage. The NSP3-UnaG fusion product was well expressed in rSA11/NSP3-FL-UnaG-infected cells, reaching levels similar to NSP3 in wildtype rSA11-infected cells. Moreover, the NSP3-UnaG protein, like functional wildtype NSP3, formed dimers in vivo. Notably, NSP3-UnaG protein was readily detected in infected cells via live cell imaging, with intensity levels ~3-fold greater than that of the NSP1-UnaG fusion product of rSA11/NSP1-FL-UnaG. Our results indicate that FP-expressing recombinant rotaviruses can be made through manipulation of the segment 7 dsRNA without deleting or interrupting any of the twelve open reading frames of the virus. Because NSP3 is expressed at levels higher than NSP1 in infected cells, rotaviruses expressing NSP3-based FPs may be a more sensitive tool for studying rotavirus biology than rotaviruses expressing NSP1-based FPs. This is the first report of a recombinant rotavirus containing a genetically engineered segment 7 dsRNA.\n\nImportancePrevious studies have generated recombinant rotaviruses that express fluorescent proteins (FPs) by inserting reporter genes into the NSP1 open reading frame (ORF) of genome segment 5. Unfortunately, NSP1 is expressed at low levels in infected cells, making viruses expressing FP-fused NSP1 less than ideal probes of rotavirus biology. Moreover, FPs were inserted into segment 5 in such a way as to compromise NSP1, an interferon antagonist affecting viral growth and pathogenesis. We have identified an alternative approach for generating rotaviruses expressing FPs, one relying on fusing the reporter gene to the NSP3 ORF of genome segment 7. This was accomplished without interrupting any of the viral ORFs, yielding recombinant viruses likely expressing the complete set of functional viral proteins. Given that NSP3 is made at moderate levels in infected cells, rotavirus encoding NSP3-based FPs should be more sensitive probes of viral infection than rotaviruses encoding NSP1-based FPs.

microbiology↗

bioRxiv2019

Genetic Polymorphisms and Molecular Mechanisms Mediating Oncolytic Potency of Reovirus Strains

The Dearing strain of Mammalian orthoreovirus (T3D) is undergoing clinical trials as an oncolytic virotherapeutic agent. In this study, a comprehensive phenotypic and genetic comparison of T3D virus stocks from various laboratories and commercial sources revealed that T3D laboratory strains differ substantially in their oncolytic activities in vitro and in vivo. Superior replication of the most-oncolytic T3D lab strain was attributed to several mechanistic advantages: virus-cell binding, viral RNA transcriptase activity, viral inclusion morphology, and differential activation of RIG-I versus NF{kappa}B-dependent signalling pathways. Viral S4, M1 and L3 gene segments were each independently associated with a distinct mechanistic advantage. Furthermore, the specific missense polymorphisms that governed replication potency were identified, and utilized to generate a hybrid of T3D laboratory strains with further-augmented replication in tumor cells. Together, the results depict an elaborate balance between reovirus replication and host-cell signaling to achieve optimal oncolytic reovirus efficacy.

microbiology↗