Biology subjects

Desai, P. J.

Publications and source records attributed to Desai, P. J..

4 recordsLinked to original sources

bioRxiv2025

m5C RNA Methylation Is Dysregulated by Oncogenic Herpesviruses via c-Myc Signaling to Counteract Host Antiviral Factors

Herpesviruses are a group of double-stranded DNA viruses known to develop versatile viral strategies to escape host immune surveillance for promoting their replication and propagation. This is illustrated by Kaposis sarcoma-associated herpesvirus (KSHV), an oncogenic gamma-herpesvirus that overcomes host immune suppression by multiple mechanisms. In this study, we reported that KSHV dysregulates 5-methylcytosine (m5C) modification and mRNA stability of host antiviral factors to benefit its lytic replication. KSHV lytic reactivation or de novo challenge led to downregulation of m5C RNA methyltransferases, NSUN2 and NSUN1 (NSUN2/1), while NSUN2/1 depletion promoted KSHV lytic replication. Such KSHV-mediated downregulation of NSUN2/1 is via suppression of the transcriptional factor c-Myc. We further performed the RNA bisulfite sequencing (RNA-BS-seq) to identify KSHV-dependent m5C modification of host mRNAs. KSHV lytic reactivation led to the significant reduction of m5C methylation and mRNA stability of TRIM25, a key activator of the RIG-I pathway, while TRIM25 depletion indeed promoted KSHV lytic replication. These host-virus interaction events were also observed in the infection of another oncogenic gamma-herpesvirus Epstein-Barr virus (EBV). Overall, our results highlighted a new strategy for human gamma-herpesviruses to counteract host antiviral factors and promote their lytic replication by manipulating host m5C RNA methylation. Significance StatementOur study has identified a novel viral mechanism of human gamma-herpesviruses to manipulate host RNA methylation machineries to subvert immune defenses and enhance viral lytic replication. In particular, our new data showed that KSHV/EBV downregulate the key 5-methylcytosine (m5C) RNA writers NSUN2/1 via c-Myc, and thus decrease m5C modification and stability of TRIM25 mRNA. As TRIM25 is a key E3 ubiquitin ligase in RIG-I signal transduction, its inhibition disrupts RIG-I mediated antiviral sensing and thus favors viral lytic replication. As human gamma-herpesviruses are critical pathogens that highly associate with multiple human diseases especially certain tumors, such studies are significant to shed light in improving the fundamental understanding of virus-host interactions and identifying new host targets for future translational applications.

microbiology↗

bioRxiv2024

The loss of both pUL16 and pUL21 in HSV-1 infected cells abolishes cytoplasmic envelopment.

Previously, we had developed synthetic genomics methods to assemble an infectious clone of herpes simplex virus type-1 (HSV-1). To do this, the genome was assembled from 11 separate cloned fragments in yeast using transformation associated recombination. The eleven fragments or "parts" spanned the 152 kb genome and recombination was achieved because of the overlapping homologous sequences between each fragment. To demonstrate the robustness of this genome assembly method for reverse genetics, we engineered different mutations that were located in distant loci on the genome and built a collection of HSV-1 genomes that contained single and different combination of mutations in 5 conserved HSV-1 genes. The five genes: UL7, UL11, UL16, UL21 and UL51 encode virion structural proteins and have varied functions in the infected cell. Each is dispensable for virus replication in cell culture, however, combinatorial analysis of deletions in the five genes revealed "synthetic-lethality" of some of the genetic mutations. Thus, it was discovered that any virus that carried a UL21 mutation in addition to the other gene was unable to replicate in Vero cells. Replication was restored in a complementing cell line that provided pUL21 in trans. One particular combination (UL16-UL21) was of interest because the proteins encoded by these genes are known to physically interact and are constituents of the tegument structure. Furthermore, their roles in HSV-1 infected cells are unclear. Both are dispensable for HSV-1 replication, however, in HSV-2 their mutation results in nuclear retention of assembled capsids. We thus characterized these viruses that carry the single and double mutant. What we discovered is that in cells where both pUL16 and pUL21 are absent, cytoplasmic capsids were evident but did not mature into enveloped particles. The capsid particles isolated from these cells showed significantly lower levels of incorporation of both VP16 and pUL37 when compared to the wild-type capsids. These data now show that of the tegument proteins, like the essential pUL36, pUL37 and VP16; the complex of pUL16 and pUL21 should be considered as important mediators of cytoplasmic maturation of the particle.

microbiology↗

bioRxiv2023

Expression and fusogenic activity of SARS CoV-2 Spike protein displayed in the HSV-1 Virion.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a zoonotic pathogen that can cause severe respiratory disease in humans. The new SARS-CoV-2 is the cause of the current global pandemic termed coronavirus disease 2019 (COVID-19) that has resulted in many millions of deaths world-wide. The virus is a member of the Betacoronavirus family, its genome is a positive strand RNA molecule that encodes for many genes which are required for virus genome replication as well as for structural proteins that are required for virion assembly and maturation. A key determinant of this virus is the Spike (S) protein embedded in the virion membrane and mediates attachment of the virus to the receptor (ACE2). This protein also is required for cell-cell fusion (syncytia) that is an important pathogenic determinant. We have developed a pseudotyped herpes simplex virus type 1 (HSV-1) recombinant virus expressing S protein in the virion envelop. This virus has also been modified to express a Venus fluorescent protein fusion to VP16, a virion protein of HSV-1. The virus expressing Spike can enter cells and generates large multi-nucleated syncytia which are evident by the Venus fluorescence. The HSV-1 recombinant virus is genetically stable and virus amplification can be easily done by infecting cells. This recombinant virus provides a reproducible platform for Spike function analysis and thus adds to the repertoire of pseudotyped viruses expressing Spike. Impact StatementThe isolation of a pseudotyped herpes simplex virus type 1 (HSV-1) virus using the Spike protein is new and innovative. This virus can be used to study entry and fusion events mediated by the S protein as well as test antibodies for their ability to neutralize this particle. In addition, these virions can be used for screening antibody specificity using the S protein displayed in its natural membrane bound conformation.

microbiology↗

bioRxiv2018

Development and Application of a High-Content Virion Display Human GPCR Array

G protein-coupled receptors (GPCRs) comprise the largest membrane protein family in humans and can respond to a wide variety of ligands and stimuli. Like other multi-pass membrane proteins, the biochemical properties of GPCRs are notoriously difficult to study because they must be embedded in lipid bilayers to maintain their native conformation and function. To enable an unbiased, high-throughput platform to profile biochemical activities of GPCRs in native conformation, we individually displayed 315 human non-odorant GPCRs (>85% coverage) in the envelope of human herpes simplex virus-1 and immobilized on glass to form a high-content Virion Display (VirD) array. Using this array, we found that 50% of the tested commercial anti-GPCR antibodies (mAbs) is ultra-specific, and that the vast majority of those VirD-GPCRs, which failed to be recognized by the commercial mAbs, could bind to their canonical ligands, indicating that they were folded correctly. Next, we used the VirD-GPCR arrays to examine binding specificity of two known peptide ligands and recovered expected interactions, as well as new off-target interactions, three of which were confirmed with real-time kinetics measurements. Finally, we explored the possibility of discovering novel pathogen targets by probing VirD-GPCR arrays with live group B Streptococcus (GBS), a common Gram-positive bacterium causing neonatal meningitis. Using cell invasion assays and a mouse model of hematogenous meningitis, we showed that inhibition of one of the five newly identified GPCRs, CysLTR1, greatly reduced GBS penetration in brain-derived endothelial cells and in mouse brains. Therefore, our work demonstrated that the VirD-GPCR array holds great potential for high-throughput, unbiased screening for small molecule drugs, affinity reagents, and deorphanization.

pharmacology and toxicology↗