Biology subjects

Majumder, K.

Publications and source records attributed to Majumder, K..

9 recordsLinked to original sources

bioRxiv2026

CTCF regulates wild-type and recombinant AAV gene expression by shaping viral chromatin

Adeno-Associated Viruses (AAVs) are powerful platforms for delivering therapeutic transgenes via recombinant AAV (rAAV) vectors. However, a limited understanding of the regulation of AAV gene expression has narrowed the ability to efficiently express therapeutic transgenes from rAAV vectors. Since rAAVs retain only the wtAAV inverted terminal repeats (ITR), we hypothesized that regulatory elements outside the ITR that govern wild-type AAV (wtAAV) gene expression can be used to modify rAAV genomes to enhance vector performance. Through in silico analysis, biochemical pulldowns, and high-throughput sequencing, we have identified that the host architectural protein CCCTC-binding Factor (CTCF) associates with the wtAAV type 2 (wtAAV2) genome but is absent from rAAV vectors. Global knockdown and site-specific deletion revealed that the CTCF binding element (CBE) on the wtAAV2 genome, located upstream of the viral P5 promoter, regulates expression of the viral Rep68/78 genes. We have re-engineered new rAAV vectors expressing a GFP reporter transgene to contain the wtAAV2-CBE upstream of the vector promoter. Our results show that CTCF binding dramatically increased rAAV transduction efficiency and GFP expression by up to four-fold across multiple cell types. This enhancement was independent of the AAV capsid serotype used for packaging rAAV vectors. CUT&RUN analysis revealed that this CBE was necessary and sufficient to regulate the chromatin landscape of wtAAV2 and rAAV2. Finally, we observed that CTCF-mediated chromatin remodeling of rAAV2 led to increased production of nascent RNA transcripts from the vector genome. Based on our findings, we propose that CTCF supports wtAAV2/rAAV gene expression by shaping the local chromatin landscape. SIMPLE ABSTRACTRecombinant Adeno-Associated Viruses (rAAV) gene therapy vectors have been engineered from wild-type AAV (wtAAV) by inserting the viral telomeres (that serve as replication and packaging signals) on either side of therapeutic transgenes. However, efficient expression of transgenes using current rAAV technologies require high doses, which can lead to sporadic toxic side effects. We hypothesized that uncharacterized regulatory elements in the wtAAV2 genome drive efficient viral gene expression and are absent from the current generation of rAAV vectors. Using in-silico analysis combined with biochemical pulldowns, high-throughput sequencing, and mutant viral systems, we have identified a novel cis-acting element bound by the cellular architectural protein CCCTC-binding Factor (CTCF). This CTCF binding element is necessary for wtAAV2 gene expression and is sufficient to enhance the rAAV vectors ability to express reporter transgenes. This CTCF-binding element regulates the chromatin landscape of the virus and its vectors. Our discovery that adding the 19-bp AAV2 CTCF-binding element enhances transgene expression without affecting vector production efficiency presents a promising new rAAV gene therapy platform that is likely to reduce clinical doses and minimize toxicity in therapeutic applications.

microbiology↗

bioRxiv2026

The interaction between virus-bound KLF4 and host-bound PARP1 directs the localization of Adeno-Associated Virus Type 2 (wtAAV2) to cellular sites of DNA damage

Wild-type adeno-associated virus type 2 (wtAAV2) is a small, non-pathogenic DNA virus within the family Parvoviridae that is modified to engineer recombinant AAV (rAAV) vectors used in gene therapy. wtAAV2 genomes localize to nuclear domains enriched in DNA Damage Response (DDR) factors through mechanisms that remain unknown. We have discovered that the host transcription factor KLF4 (Kruppel-like Factor 4) and the DDR protein PARP1 (poly-ADP ribose polymerase 1) are key effectors regulating wtAAV2 nuclear trafficking. In-silico analysis revealed that wtAAV2-associated genomic sites are enriched in KLF4 binding motifs. Confocal imaging demonstrated a close spatial association between KLF4 and wtAAV2 nuclear reservoirs, consistent with KLF4s known ability to form nuclear condensates. Mutation of the KLF4 binding site within the wtAAV2 genome and RNAi-mediated knockdown of KLF4 globally reduced the expression of viral Rep68/78 genes and attenuated the localization of the viral genome to cellular DDR sites. Chemical inhibition of the KLF4-interacting protein PARP1 with Olaparib decreased the ability of wtAAV2 genomes to localize to cellular DDR sites and transcribe viral genes. Ectopic expression of wild-type PARP1, but not its KLF4-binding-deficient mutant, rescued wtAAV2 gene expression in PARP1-deficient cells. These findings define a novel mechanism by which wtAAV2 exploits the interaction between host transcription factors and DNA repair machinery to establish a persistent nuclear niche. Insertion of KLF4-binding elements into recombinant AAV2 (rAAV2) gene therapy vectors is sufficient to enhance transduction of target cells, providing a framework for engineering vectors with improved nuclear targeting and transcriptional activity. IMPORTANCEWild-type Adeno-associated virus type 2 (wtAAV2) has emerged as the preferred platform for engineering gene therapy vectors due to its non-pathogenic nature and ability to persist in host cells long-term. However, limited understanding of how wtAAV2 genomes navigate the nuclear environment to establish viral reservoirs has hindered the development of efficient recombinant AAV (rAAV) vectors. We demonstrate that the protein KLF4 bound to wtAAV2 genome recruits the virus to cellular KLF4 sites bound by PARP1. Disruption of either KLF4 binding, PARP1 activity or KLF4-PARP1 interaction significantly impairs wtAAV2 localization and transcription, highlighting the importance of their function in the non-replicative wtAAV2 life cycle. KLF4 binding sites are sufficient to improve the expression of transgenes from rAAV vectors and increase their association with cellular DDR proteins. This study advances our understanding of wtAAV2-host interactions and opens new avenues for improving rAAV gene therapy platforms.

microbiology↗

bioRxiv2025

Adeno-Associated Virus 2 (AAV2) - induced RPA exhaustion generates cellular DNA damage and restricts viral gene expression

Parvoviruses are single-stranded DNA viruses that have been modified to serve as vehicles for therapeutic transgene delivery in the form of recombinant Adeno-Associated Virus (rAAV2) vectors or rodent parvovirus-derived oncolytic agents. Infection with viruses of the Parvoviridae family induces a cellular DNA Damage Response (DDR) signal that supports virus replication. However, it remains unknown whether rAAV2 vectors or non-replicative AAV2 genomes induce cellular DDR signals, which might be deleterious to the cell. To determine the impact of AAV2/rAAV2 genomes on the integrity of the host chromosome, we have pulsed AAV2/rAAV2 infected cells with BrdU analogs followed by single-molecule imaging of the cellular replisomes and proteomic analysis of the host replication forks. We discovered that non-replicative AAV2/rAAV2 genomes are sufficient to induce replication stress on the host genome, leading to DDR signals in a dose-dependent manner. Moreover, infection with replication-competent AAV2 leads to enrichment of replication stress proteins, DNA repair factors and RNA processing machinery on cellular replication forks. However, neither the AAV2 Inverted Terminal Repeats (ITRs) that are retained in rAAV2s nor empty capsids are sufficient to induce host-cell replication stress. Strikingly, incoming AAV2 genomes associate with the single-stranded DNA binding protein RPA in host cells in a dose-dependent manner, progressively shortening cellular replication forks. These elevated levels of AAV2-induced cellular replication stress eventually leads to accumulation of DDR signals in the nucleus. Chemical inhibition of RPA activity and RNAi-mediated knockdown leads to de-repression of the AAV2 genome, increasing Rep 68/78 gene expression. Ectopic expression of RPA rescues AAV2-induced replication stress. Taken together, our findings suggest that depletion of cellular stores of RPA molecules by competing AAV2 genomes restrict viral gene expression and cause cellular DNA damage. AUTHOR SUMMARYAdeno-Associated Viruses 2 (AAV2) are modified to design therapeutic gene therapy vectors, but how they interact with the guardians of host DNA remains unknown. In this work, we show that AAV2 genomes compete with the host cell for the single-stranded DNA binding protein RPA, rendering the host vulnerable to replication stress leading to both suppression of the viral gene expression and induction of cellular DNA breaks. These findings provide insights into how gene therapies delivered at high doses could have genotoxic effects, underscoring the importance of engineering AAV2-based gene therapy platforms that express efficiently at lower doses.

microbiology↗

bioRxiv2025

Human Papillomavirus Genomes Associate with Active Host Chromatin during Persistent Viral Infection

Human papillomaviruses (HPVs) maintain their genomes as minichromosomes in the nuclei of infected keratinocytes. This study investigates the association of HPV31 genomes with host chromatin using both HiC and 4C-seq chromosome conformation capture techniques. We show that HPV31 genomes preferentially associate with transcriptionally active A compartments of host chromatin, regions of open chromatin defined by ATAC-seq, and super-enhancers defined by Brd4 and H3K27ac ChIP-seq. The viral genome association sites were also highly correlated with genomic loci previously identified as common HPV integration sites in cervical cancers. Recent studies have shown that transcriptionally active sites are prone to dsDNA breaks, and we find a strong correlation among dsBREAK datasets with transcriptionally active and open regions of host chromatin and the HPV31 genome association sites defined in our study. These findings suggest that HPV genomes associate with cellular transcriptional epicenters to maintain active viral gene expression during persistent infection, but also indicate that the susceptibility of these regions to dsDNA breaks could explain their propensity for viral DNA integration in HPV-associated cancers.

genomics↗

bioRxiv2024

USP1/UAF1 targets polyubiquitinated PCNA with an exo-cleavage mechanism that enriches for monoubiquitinated PCNA

DNA damage tolerance (DDT) is an important pathway that allows our cells to bypass DNA lesions during replication. DDT is orchestrated by ubiquitination of PCNA: Monoubiquitination (PCNA-Ub) initiates recruitment of TLS polymerases but also serves as substrate for K63-linked polyubiquitination that leads to HR-mediated bypass mechanisms. Recent work on USP1/UAF1 inhibition revealed that K48-linked chains are also formed on PCNA, resulting in its proteasomal degradation. USP1/UAF1 is established as deubiquitinating enzyme (DUB) for PCNA-Ub, but little is known about deubiquitination of chains on PCNA. Here we show that USP1/UAF1 cleaves both K48 and K63-linked ubiquitin chains on PCNA efficiently, using an exo-cleavage mechanism. Kinetic analysis reveals that USP1/UAF1 prefers cleaving the ubiquitin-ubiquitin bond over cleavage of the ubiquitin-PCNA isopeptide bond and therefore treats poly- and monoubiquitinated PCNA as different substrates. A cryo-EM structure of USP1/UAF1 with a K63-diubiquitin and structure-based mutagenesis reveals that its mechanistic preference is maintained in evolution. Its kinetic mechanism results in relative enrichment of monoubiquitinated PCNA that could initially promote TLS over HR-like bypass. Taken together, these results suggest that USP1/UAF1 could be important in temporary protection of PCNA against K48- and K63-linked polyubiquitination and highlight this DUB as a potential regulator of DDT pathway choice.

biochemistry↗

bioRxiv2024

Hepatitis B Virus genomes associate with cellular sites of DNA damage by inducing replication stress

Hepatitis B Virus (HBV) is a leading cause of liver cancer, with almost 300 million infected individuals worldwide. Although HBV-infected patients benefit from drug regimens that help to control chronic infection, they are rarely clinically cured of HBV. The HBV genome persists in the nucleus of infected hepatocytes in the form of a covalently closed circular DNA (cccDNA) molecule, a reservoir of HBV DNA molecules that serve as the template for reactivation of long-term chronic HBV. However, despite playing a central role in the viral life cycle, little is understood about where cccDNA molecules localize, why they are so stable, and how they impact the host nuclear compartment. Perhaps because of this, there are few treatments that target cccDNA, which is critical for eradication of clinical HBV. Here, we show that HBV infection induces a cellular DNA Damage Response (DDR) that is comparable with cells undergoing replication stress, and this cellular replication stress is initiated after the formation of cellular cccDNA molecules. Using a novel high-throughput chromosome conformation capture technology that monitors the localization of HBV cccDNA molecules, we show that cccDNA molecules persist in the vicinity of many cellular fragile sites. Induction of cellular DNA damage leads to relocalization of the viral HBx oncoprotein to DDR sites in an ATM, ATR and DNA-PK dependent manner. Our findings contribute to the understanding of how HBV cccDNA navigates the host nuclear environment, identifying functional targets for development of therapies against HBV infection and resulting liver cancer. ImportanceHepatitis B Virus (HBV) is the leading infectious cause of liver cancer globally. The virus persists in the nucleus long term by forming reservoirs in human liver cells. We have discovered that the HBV DNA localizes to sites on the host genome associated with DNA damage, and in doing so, HBV interferes with the hosts ability to efficiently amplify itself. This results in the induction of cellular DNA breaks, which we propose contributes to eventual cancer progression. Our findings provide new insights into how HBV infection may lead to liver cancer.

microbiology↗

bioRxiv2023

Genomes of the Autonomous Parvovirus Minute Virus of Mice Induce Replication Stress Through RPA Exhaustion

The oncolytic autonomous parvovirus Minute Virus of Mice (MVM) establishes infection in the nuclear environment by usurping host DNA Damage Response (DDRs) in the vicinity of cellular DNA break sites. MVM replication induces a global cellular DDR that is dependent on signaling by the ATM kinase and inactivates the cellular ATR-kinase pathway. However, the mechanism of how MVM generates cellular DNA breaks remains unknown. Using single molecule DNA Fiber Analysis, we have discovered that MVM infection leads to a shortening of host replication forks as infection progresses, as well as induction of replication stress prior to the initiation of virus replication. Ectopically expressed viral non-structural proteins NS1 and NS2 are sufficient to cause host-cell replication stress, as is the presence of UV-inactivated non-replicative MVM genomes. The host single-stranded DNA binding protein Replication Protein A (RPA) associates with the UV-inactivated MVM genomes, suggesting MVM genomes might serve as a sink for cellular stores of RPA. Overexpressing RPA in host cells prior to UV-MVM infection rescues DNA fiber lengths and increases MVM replication, confirming that MVM genomes deplete RPA stores to cause replication stress. Together, these results indicate that the presence of ssDNA in the nucleus generated by MVM genomes and viral proteins induces replication stress in the host cell through RPA exhaustion, rendering the host genome vulnerable to additional DNA breaks. AUTHOR SUMMARYParvoviruses are used in the clinic to design recombinant gene therapy vectors and as oncolytic agents. The autonomous parvovirus MVM utilizes the host cells DNA damage response machinery to replicate in host cells and cause additional DNA breaks. However, the mechanism of MVM-induced DNA damage remains unknown. We have discovered that MVM sequesters the host DNA repair protein RPA, which normally associates with single stranded DNA in the nucleus, rendering the host genome susceptible to replication stress. Our study provides insights into the mechanisms utilized by single-stranded DNA viruses to amplify host-cell DNA damage.

microbiology↗

bioRxiv2022

Denaturation resistant P2 tetramer imports fatty acids from human serum into intraerythrocytic Plasmodium falciparum

The initiation of asymmetric karyokinesis of intraerythrocytic Plasmodium falciparum (Pf) begins without dismantling the nuclear envelope showing the hallmark feature of closed mitosis (1-10). In Pf, karyokinesis precedes cytokinesis and cell body formation (6, 8-10). Regulation at the beginning of nuclear division either through checkpoints or by importing serum components was largely unknown. At the trophozoite stage, PfP2 tetramer trafficked to the infected erythrocyte (IE) surface and the inaccessibility of IE surface PfP2 to its bonafide ligand led to the arrest of nuclear division (11-13). Here we show that PfP2 tetramer localization on the IE surface and the beginning of nuclear division are concomitant in nature. Synthetically induced denaturation resistant PfP2 tetramer interacts with human serum fatty acids and phospholipids for its import into IEs at the beginning of karyokinesis. In the natively folded denaturation resistant PfP2 tetramer cage, the Cys-Cys redox switch regulates the binding and subsequent release of fatty acids on the IE surface. This mechanistic insight of fatty acids import inside IEs using synthetically induced denaturation resistant PfP2 tetramer provides an unique drug screening platform for novel small molecule screening against malaria.

molecular biology↗

bioRxiv2020

Immuno-informatics approach for multi-epitope vaccine designing against SARS-CoV-2

The novel Corona Virus Disease 2019 (COVID-19) pandemic has set the fatality rates ablaze across the world. So, to combat this disease, we have designed a multi-epitope vaccine from various proteins of Severe Acute Respiratory Syndrome Corona virus 2 (SARS-CoV-2) with an immuno-informatics approach, validated in silico to be stable, non-allergic and antigenic. Cytotoxic T-cell, helper T-cell, and B-cell epitopes were computationally predicted from six conserved protein sequences among four viral strains isolated across the world. The T-cell epitopes, overlapping with the B-cell epitopes, were included in the vaccine construct to assure the humoral and cell-mediated immune response. The beta-subunit of cholera toxin was added as an adjuvant at the N-terminal of the construct to increase immunogenicity. Interferon-gamma inducing epitopes were even predicted in the vaccine. Molecular docking and binding energetics studies revealed strong interactions of the vaccine with immune-stimulatory toll-like receptors (TLR) -2, 3, 4. Molecular dynamics simulation of the vaccine ensured in vivo stability in the biological system. The immune simulation of vaccine evinced elevated immune response. The efficient translation of the vaccine in an expression vector was assured utilizing in silico cloning approach. Certainly, such a vaccine construct could reliably be effective against COVID-19.

bioinformatics↗