Biology subjects

Savanagouder, M.

Publications and source records attributed to Savanagouder, M..

3 recordsLinked to original sources

bioRxiv2026

Host DNA repair factors empower a mechanism of antiviral nucleoside analog resistance

How host functions affect resistance to antiviral drugs is poorly understood. Ganciclovir, a chain-terminating nucleoside analog, is a first-line therapy against human cytomegalovirus, a widespread herpesvirus that causes life-threatening disease in immunocompromised individuals and newborns. Ganciclovir resistance, which is caused by mutations that affect the viral kinase, UL97 and/or the viral polymerase, UL54, can cause treatment failures. Among these mutations, those reducing the exonuclease activity of the viral DNA polymerase permit ganciclovir incorporation without chain termination. However, the fate of DNA strands containing the incorporated nucleotide analog is unknown. We show here that template DNA containing ganciclovir fails to support DNA synthesis of the complementary strand by exonuclease-mutant polymerase. Moreover, while DNA synthesis and ganciclovir incorporation are limited in drug-treated fibroblasts infected by virus with wild-type polymerase, an exonuclease-resistant mutant virus can better synthesize full-length genomes and incorporate substantially more ganciclovir into DNA. Notably, ganciclovir is lost from DNA when drug is removed, suggesting that ganciclovir-containing templates are repaired. We identify the host nucleotide excision repair component, XPA, and the repair enzyme, polymerase kappa, as each being necessary for mutant virus ganciclovir resistance and polymerase kappa as being required for the mutants cidofovir resistance, demonstrating a role for host DNA repair machinery in a mechanism of antiviral resistance. We propose a model for this mechanism, which has relevance for at least one other antiviral drug and likely other nucleoside analog therapeutics, and highlights the participation of host DNA repair machinery during human cytomegalovirus DNA replication. IMPORTANCENucleoside analogues such as ganciclovir, which is a leading drug for preventing and treating human cytomegalovirus, are a critical defense against viral diseases, but antiviral resistance often results in treatment failures. This study reveals a critical role for host DNA repair in a mechanism of resistance to ganciclovir, and identifies at least one specific repair pathway that permits viral DNA synthesis in the presence of ganciclovir, defining a mechanism by which cellular DNA repair pathways conspire to enable antiviral drug resistance. This mechanism is relevant to at least one other antiviral drug and may apply to other antiviral and anticancer agents. The study also showcases the participation of host DNA repair machinery during human cytomegalovirus DNA synthesis.

microbiology↗

bioRxiv2025

Chromosome-associated spot formation by human cytomegalovirus immediate early 1 (IE1) protein

HCMV is associated with severe disease in immunocompromised patients and is a cause of congenital disease. Already more than 30 years ago, IE1 was described to bind to mitotic chromosomes, however the implications of this finding have yet to be determined. HCMV can infect a wide range of cell types and was also detected in different tumours; however, the majority of molecular studies relating to this virus are performed in fibroblasts or monocytic cells. We examined the localization of HCMV IE1 across multiple cell types, including tumour cell lines, to better understand how the protein behaves in these cellular contexts. IE1 is one of the first HCMV genes expressed after lytic infection of the cell and it was observed to distribute evenly over the chromosomes in so-called "chromosome painting" pattern. In leukemia and glioblastoma cells we detected a novel mitotic localization pattern of IE1 - in chromosome-associated spots (CAS), in addition to "painting the chromosomes", as reported before. We demonstrate that the IE1 core domain mediates CAS localization and that the distribution pattern of IE1 on mitotic chromosomes is influenced by both the cell type being infected and the level of IE1 expression. The novel CAS localization of IE1 suggests a new, possibly cell-type specific function of this protein, distinct from its role in transcriptional regulation.

microbiology↗

bioRxiv2023

Dissecting the Ca2+ dependence of Mycobacterium tuberculosis DesA1 function

Mycobacterium tuberculosis (M. tb) has a complex cell wall, largely composed of mycolic acids and long-chain fatty acids that play a crucial role in maintaining its integrity and permeability. This complex lipid structure has a role in abrogating the process of phagosome-lysosome fusion and infection establishment. The M. tb desaturase A1 (DesA1) catalyzes the introduction of position-specific double bonds, a key step in the biosynthesis of a diverse range of mycolic acids. We have previously demonstrated that M. tb DesA1 is a Ca2+-binding protein, belonging to the extended {beta}{gamma}-crystallin superfamily. Using a combination of biophysical and genetic approaches, we investigated the structural and functional significance of Ca2+ binding on DesA1 activity. A protein unfolding assay of the protein in the presence and absence of Ca2+ shows that Ca2+ binding imparts structural stability to DesA1. To identify the role of Ca2+, we introduced mutations at key residues in the identified Ca2+-binding motif of DesA1 and generated F303A, E304Q, and F303A-E304Q variants of DesA1. We identified F303 as a hot point which disables the protein for Ca2+ binding. Two other mutations E304Q and F303A-E304Q showed reduced Ca2+ binding. Complementation of a conditionally complemented desA1 deletion mutant strain of Mycobacterium smegmatis with these mutants, either failed to complement its growth phenotype or led to a compromise in complementation. In addition, the F303A and F303A-E304Q complements exhibit increased sensitivity to isoniazid, a first-line anti-tubercular drug, pointing to a cell wall permeability defect in these strains. Our findings highlight the critical importance of Ca2+ in the functioning of DesA1 and its implicit role in the maintenance of mycobacterial cellular integrity.

microbiology↗