Varghese, G. R. | Search bioRxiv

bioRxiv2025

Reproducible generation of Nipah virus pseudovirions with uniform expression of F and G surface glycoproteins for high-throughput neutralization assays

Nipah virus (NiV) is a pathogen to be handled in BSL-4 facilities. Multiple surrogate systems such as virus-like particles (VLPs) and pseudoviruses enable carrying out NiV neutralization assays and study of virus entry pathways in biosafety level-2 (BSL-2) facilities. These are dual protein expression and surface display systems comprising NiV structural glycoproteins F and G as the key components. During generation of NiV VLPs or pseudovirions, ensuring batch to batch uniformity is a major concern due to the lack of proportionate expression of these proteins in the producer cells as well as their consistent incorporation in the particles. We established HEK293 pseudovirion producer cells that stably co-express NiV F and G proteins to address the issue. Fluorescence-activated cell sorting (FACS) analysis of clonally selected cells for high and uniform level F and G protein co-expression; and their further expansion were carried out to further refine the system. High titer vesicular stomatitis virus (VSV)-based pseudoviruses which showed consistent expression of both the glycoprotein were reproducibly generated from these producer cells. In functional assays, these pseudovirions exhibited a dose-dependent neutralization by commercial anti-NiV F and G antibodies as well as by convalescent serum from Nipah recovered patients. A pseudovirus neutralization test (PVNT) with a secreted alkaline phosphatase (SEAP) as the reporter was established in the study. The assay supports high-throughput adaptability with a quick turn-around time. It will aid large-scale human and animal serosurveillance studies in Nipah endemic regions as well as screening of virus entry inhibitors and monoclonal antibodies.

microbiology↗

bioRxiv2022

BRCA1 Hypermethylation In Sporadic Breast Cancers: Discovering A Novel Pathway To Tumorigenesis Via Coordinate NBR2 Deregulation And TNBC Transformation

Women with a family history of mutations in the Breast cancer susceptibility gene, BRCA1 will have an increased risk of developing breast neoplasms. However, majority of the breast cancers are sporadic where BRCA1 mutations are very rare. Instead, 5-65% of sporadic cases manifest BRCA1 promoter hypermethylation and 30-40% of such cases develop into Triple Negative Breast Cancers. Even then, the molecular mechanism of BRCA1 hypermethylation mediated breast tumorigenesis has remained an enigma till date. Here, we present a novel tumorigenesis pathway for breast cancers that engenders from BRCA1 hypermethylation by generating site-specific methylations in the BRCA1 promoter using a modified version of CRISPR technology. We report that induction of site-specific methylation on BRCA1 promoter effectuates a downregulation in BRCA1 expression via alteration in the balance between its alternate transcripts {beta} and . Induced BRCA1 hypermethylation is also responsible for the attenuation of a long noncoding RNA, NBR2 (Neighbour of BRCA1 gene 2), which is transcribed through the bidirectional BRCA1 promoter in the reverse direction. Downregulation of NBR2 activates a feedback loop by leading to further downregulation of BRCA1 which is more evident under glucose starvation conditions and is associated with impaired DNA damage repair. BRCA1 hypermethylation also results in significant overexpression of {beta}-hCG (human chorionic gonadotrophin), which was found to be associated with highly aggressive and drug-resistant forms of BRCA1 mutated breast cancers invitro & in vivo in our previous study. Further, we report a change in the hormone receptor levels as the tumor progresses which demonstrates how BRCA1 deficient cells modulate their expression of ER- and ER-{beta} to promote their proliferation in early stages of tumor development and at later stages, transform to a basal tumor subtype by shedding down the expression of ER- & PR. Interestingly, we also discovered that modulation of ER- expression upon BRCA1 hypermethylation is responsible for the alteration in BRCA1 transcript ratio. Finally, in in vivo mouse studies, BRCA1 hypermethylated tumors were found to be much larger, aggressive and invasive as compared to wildtype, BRCA1 and NBR2 knockdown tumors with downregulation of ER- and PR; which explains the most probable reason behind high relapse rates in BRCA1 hypermethylated tumors. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/490082v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1c81155org.highwire.dtl.DTLVardef@1ea1fdcorg.highwire.dtl.DTLVardef@1d1b5e9org.highwire.dtl.DTLVardef@fddd22_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗