Biology subjects

Lodha, M.

Publications and source records attributed to Lodha, M..

8 recordsLinked to original sources

bioRxiv2026

Genetic innovation in coronaviruses driven by a viral nuclease

Genetic variation in viruses is well known to arise from polymerase-driven nucleotide misincorporation. However, insertion and deletion (indel) mutations that occur at lower, largely unknown, frequencies can underly more dramatic phenotypic changes that emerge when advantageous. Using a human coronavirus (HCoV-OC43) construct that reports rare indel mutations, we show that non-structural protein-15 (NSP15), a nuclease encoded by coronaviruses, can drive the acquisition of a class of insertion mutations. Ultra-deep sequencing of both HCoV-OC43 and SARS-CoV-2 populations reveals a similar requirement for NSP15 during insertion mutant generation. Overall, the insertional mutation frequency exceeded 10-3/genome in these two coronaviruses. Analysis of thousands of HCoV-OC43 and SARS-CoV-2 insertion mutants reveals a mutational process in which NSP15 cuts viral RNA, yielding oligonucleotides that correspond to inserts that are acquired at distal genomic locations. The presence of an insertion mutation at the S1/S2 junction in the SARS-CoV-2 spike protein that generates a furin cleavage site and enhances viral transmissibility, may have been necessary for enabling the COVID19 pandemic. We found numerous examples of potential furin cleavage site acquisition and replacement through insertion mutation during the normal course of coronavirus replication. Such events are, therefore, likely commonplace in coronavirus populations of a size that occurs in nature.

microbiology↗

bioRxiv2026

Interferon-gamma induced RNF213 targets nascent double membrane vesicles to inhibit coronavirus primary translation

Innate immune systems employ many mechanisms to inhibit the replication of viruses that profoundly affect host range and pathogenicity. To identify proteins that restrict human respiratory virus replication, we executed CRISPR screens targeting both type-I and type-II interferon stimulated genes. Among the candidate antiviral genes identified, the giant ring finger protein 213 (RNF213) was a key mediator of antiviral activity of interferon gamma (IFN{gamma}) against the human betacoronavirus HCoV-OC43. RNF213 has ATPase and ubiquitin ligase activities, both of which were required for anti-HCoV-OC43 activity. Using newly designed reporter viruses, we demonstrate that RNF213 acts by a previously undescribed mechanism, inhibiting the translation of incoming viral genomes. RNF213 colocalized and coimmunoprecipitated with the primary viral translation product, NSP3, that orchestrates the formation coronavirus replication compartments. Notably, RNF213 restricted genetically diverse common human coronaviruses, but SARS-CoV-2 apparently evades interaction with and inhibition by RNF213.

microbiology↗

bioRxiv2025

Murine cytomegalovirus evolved a cell-cycle regulator (m54.5) within the highly conserved viral DNA polymerase gene

Ribosome profiling (Ribo-seq) coupled with transcription start site profiling time-course analyses recently unveiled hundreds of novel viral gene products in lytic murine cytomegalovirus (MCMV) infection. One of these is the m54.5 open reading frame (ORF) located within the highly conserved viral DNA polymerase locus (M54). Interestingly, the m54.5 ORF is expressed from its own transcript (m54.5 RNA) with early gene expression kinetics, and at much higher levels than M54. In this study, we show that m54.5 encodes a nuclear viral protein (m54.5p) that contributes to cell cycle regulation during lytic MCMV infection. We show that m54.5p interacts with components of the anaphase-promoting complex/cyclosome (APC/C) and the phosphatase-6 (PP6) complex. Nocodazole mitotic arrest assays confirmed G1 cell cycle arrest and dysregulation by m54.5. Serum starvation revealed impaired cell cycle progression to S-phase. Notably, m54.5p is not conserved in other cytomegaloviruses but functionally mimics the UL21a protein of human cytomegalovirus (HCMV), which similarly targets the master cell cycle regulator APC/C to disrupt cell cycle progression. m54.5 thus represents convergent evolution to HCMV UL21a in MCMV within the highly conserved viral DNA polymerase gene. Nevertheless, we found that m54.5p is dispensable for viral replication in cultured mouse fibroblasts, indicative of redundant cell cycle regulation in lytic MCMV infection. These findings highlight a surprising genomic plasticity of herpesviruses, facilitating the evolution of an independent transcript encoding for a >200 aa gene product within a deeply conserved viral gene locus. Author SummarySystems biology approaches have revealed a surprising complexity of herpesvirus gene products. Using advanced sequencing approaches, we discovered a novel gene, m54.5, that independently evolved within a highly conserved region of the murine cytomegalovirus (MCMV) genome. This gene, which shows no conservation in other CMVs, produces a nuclear protein, m54.5p, abundantly expressed early during infection. We show that m54.5p interacts with host cell cycle regulators--the anaphase-promoting complex/cyclosome (APC/C) and phosphatase-6 (PP6)--to arrest cells in G1 phase and block progression into S phase. This function and underlying mechanism are reminiscent of the unrelated UL21a protein in human cytomegalovirus, illustrating how distinct viruses can evolve similar strategies to control host cell division. Despite its role in cell cycle disruption, m54.5p is not required for MCMV replication in cultured cells, suggesting redundant viral mechanisms. Our findings reveal an unexpected plasticity of herpesvirus genomes to evolve new, functional transcripts and proteins even within one of the most highly conserved genomic regions. Our findings thereby reshape our understanding of herpesvirus evolution and virus-host interaction.

microbiology↗

bioRxiv2024

Identifying Modulators of Cellular Responses by Heterogeneity-sequencing

The response of individual cells to drug treatment, virus infections or other molecular stimuli is highly heterogeneous and depends on the cells initial state. Library preparation for single-cell transcriptomics is destructive, precluding a direct comparison between the initial state and the stimulus outcome. Consequently, current methods are restricted to identifying correlative associations rather than resolving causal drivers of heterogeneous outcomes. We developed Heterogeneity-seq, which combines single-cell RNA-seq with metabolic RNA labeling (scSLAM-seq) and double machine learning to overcome this limitation. By leveraging simultaneous measurements of unlabeled and labeled RNA in individual cells, Heterogeneity-seq uncovers the transition from pre-stimulated cell states to distinct stimulation outcomes across thousands of cells. These links enable the identification of factors that causally govern heterogeneous cellular responses. We used Heterogeneity-seq to identify both known and novel genes that drive responses to drug treatment, as well as pro- and antiviral host factors governing cytomegalovirus infection.

bioinformatics↗

bioRxiv2023

Correcting 4sU induced quantification bias in nucleotide conversion RNA-seq data

Nucleoside analogues like 4-thiouridine (4sU) are used to metabolically label newly synthesized RNA. Chemical conversion of 4sU before sequencing induces T-to-C mismatches in reads sequenced from labelled RNA, allowing to obtain total and labelled RNA expression profiles from a single sequencing library. Cytotoxicity due to extended periods of labelling or high 4sU concentrations has been described, but the effects of extensive 4sU labelling on expression estimates from nucleotide conversion RNA-seq have not been studied. Here, we performed nucleotide conversion RNA-seq with escalating doses of 4sU with short-term labelling (1h) and over a progressive time course (up to 2h) in different cell lines. With high concentrations or at later time points, expression estimates were biased in an RNA half-life dependent manner. We show that bias arose by a combination of reduced mappability of reads carrying multiple conversions, and a global, unspecific underrepresentation of labelled RNA due to impaired reverse transcription efficiency and potentially global reduction of RNA synthesis. We developed a computational tool to rescue unmappable reads, which performed favourably compared to previous read mappers, and a statistical method, which could fully remove remaining bias. All methods developed here are freely available as part of our GRAND-SLAM pipeline and grandR package.

bioinformatics↗

bioRxiv2022

Decoding murine cytomegalovirus

The genomes of both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) were first sequenced over 20 years ago. Similar to HCMV, the MCMV genome had initially been proposed to harbor {approx}170 open reading frames (ORFs). More recently, omics approaches revealed HCMV gene expression to be substantially more complex comprising several hundreds of translated ORFs. Here, we provide a state-of-the art reannotation of lytic MCMV gene expression based on integrative analysis of a large set of omics data. Our data reveal 363 viral transcription start sites (TiSS) that give rise to 380 and 454 viral transcripts and ORFs, respectively. The latter include >200 small ORFs, some of which represented the most highly expressed viral gene products. By combining TiSS profiling with metabolic RNA labelling and chemical nucleotide conversion sequencing (dSLAM-seq), we provide a detailed picture of the expression kinetics of viral transcription. This not only resulted in the identification of a novel MCMV immediate early transcript encoding the m166.5 ORF, which we termed ie4, but also revealed a group of well-expressed viral transcripts that are induced later than canonical true late genes and contain an initiator element (Inr) but no TATA- or TATT-box in their core promoters. We show that viral uORFs tune gene expression of longer viral ORFs expressed in cis at translational level. Finally, we identify a truncated isoform of the viral NK-cell immune evasin m145 arising from a viral TiSS downstream of the canonical m145 mRNA. Despite being {approx}5-fold more abundantly expressed than the canonical m145 protein it was not required for downregulating the NK cell ligand, MULT-I. In summary, our work will pave the way for future mechanistic studies on previously unknown cytomegalovirus gene products in an important virus animal model. Author summaryWe conducted a comprehensive characterization and reannotation of murine cytomegalovirus (MCMV) gene expression during lytic infection in murine fibroblasts using an integrative multi-omics approach. This unveiled hundreds of novel transcripts that explained the expression of close to 300 so far unknown viral open reading frames (ORFs). Interestingly, small viral ORFs (sORFs) were amongst the most highly expressed viral gene products and thus presumably encode for important viral microproteins of unknown function. However, we also show that sORFs located upstream of larger ORFs tune the expression of the downstream ORFs at the level of translation. We classified viral transcription start sites (TiSS) based on their expression kinetics obtained by a new combination of metabolic RNA labelling with transcription start sites profiling. This not only identified a so far unknown viral immediate-early transcript (ie4, m166.5 RNA) but also revealed a novel class of viral late transcripts that are expressed later than canonical true late genes and lack TATA box-like motifs. We exemplify for the m145 locus how so far unknown TiSS give rise to abundantly expressed truncated viral proteins. In summary, we provide a state-of-the-art annotation of an important model virus, which will be instrumental for future studies on CMV biology, immunology and pathogenesis.

microbiology↗

bioRxiv2022

Multi-omics reveals principles of gene regulation and pervasive non-productive transcription in the human cytomegalovirus genome

For decades, human cytomegalovirus (HCMV) was thought to express {approx}200 viral proteins during lytic infection. In recent years, systems biology approaches uncovered hundreds of additional viral gene products and suggested thousands of viral sites of transcription initiation. Despite all available data, the molecular mechanisms of HCMV gene regulation remain poorly understood. Here, we provide a unifying model of productive HCMV gene expression employing transcription start site profiling combined with metabolic RNA labeling as well as integrative computational analysis of previously published big data. This approach defined the expression of >2,600 high confidence viral transcripts and explained the complex kinetics of viral protein expression by cumulative effects of translation of incoming virion-associated RNA, multiple transcription start sites with distinct kinetics per viral open reading frame, and differences in viral protein stability. Most importantly, we identify pervasive transcription of transient RNAs as a common feature of this large DNA virus with its human host.

microbiology↗

bioRxiv2021

Th17 cell master transcription factor RORC2 regulates HIV-1 gene expression and viral outgrowth

Among CD4+ T-cells, T helper 17 (Th17) cells are particularly susceptible to HIV-1 infection and are depleted from mucosal sites, which causes damage to the gut barrier resulting in microbial translocation-induced systemic inflammation, a hallmark of disease progression. Furthermore, a proportion of latently infected Th17 cells persist long-term in the gastro-intestinal lymphatic tract, where low-level HIV-1 transcription is observed. This residual viremia contributes to chronic immune activation. Thus, Th17 cells are key players in HIV pathogenesis and viral persistence, however it is unclear why these cells are highly susceptible to HIV-1 infection. Th17 cell differentiation depends on expression of the master transcriptional regulator RORC2, a retinoic acid-related nuclear hormone receptor that regulates specific transcriptional programs by binding to promoter/enhancer DNA. Here, we report that RORC2 is a key host-cofactor for HIV replication in Th17 cells. We found that specific inhibitors that bind to the RORC2 ligand-binding domain reduced HIV replication in CD4+ T-cells. Depletion of RORC2 inhibited HIV-1 infection, whereas RORC2 overexpression enhanced it. RORC2 was found to promote HIV-1 gene expression. Chromatin immune precipitation revealed that RORC2 binds to the nuclear receptor responsive element (NRRE) in the HIV-1 LTR. In treated HIV-1 patients, RORC2+ CD4 T cells contained more proviral DNA than RORC2- cells. Pharmacological inhibition of RORC2 potently reduced HIV-1 outgrowth in CD4+ T-cells from antiretroviral-treated patients. Altogether, these results provide a new explanation as to why Th17 cells are highly susceptible to HIV-1 infection and point to RORC2 as a cell-specific target for HIV-1 therapy. Significance statementHIV-1 infects CD4 T cells and among these, Th17 cells are known to be particularly permissive for virus replication. Infection of Th17 cells is critical for AIDS pathogenesis and viral persistence, however it is not clear why these cells are highly permissive to HIV-1. We found that Th17 cell permissiveness depends on expression of the hormone receptor RORC2, which is the master transcriptional regulator of Th17 cell differentiation. We identify RORC2 as a new, cell-specific host-dependency factor that can be targeted by small molecules. Our results point to RORC2 as a cell-specific target for HIV-1 therapy, an entirely new concept in the field, and suggest HIV-1 might have evolved to exploit RORC2 to promote its own persistence.

microbiology↗