Biology subjects

Zwolinska, K.

Publications and source records attributed to Zwolinska, K..

4 recordsLinked to original sources

bioRxiv2026

Preferential recruitment of PML isoform II by incoming HPV16 genome transport vesicles facilitates early stage of infection

The interplay between promyelocytic leukemia nuclear bodies (PML NBs) and viral infections is complex and multifaceted, with viruses often engaging in a delicate balance of exploitation and evasion of host immune responses. It is well-established that incoming human papillomavirus (HPV) virions associate with PML NBs; however, the mechanism by which HPV targets PML NBs to facilitate early transcription and replication remains elusive. We have shown that HPV genomes are delivered to the nucleus inside membrane-bound vesicles during mitosis and that PML proteins are recruited to HPV-harboring nuclear vesicles prior to egress. Herein, we demonstrate that the PML isoform II (PMLII), but not other PML isoforms, is selectively attracted to incoming HPV still residing in transport vesicles. Using individual PML NB isoform and HPV16 minor protein, L2, over-expression experiments in HeLa cells, we conclude that PML II binds to the lipids of the HPV harbouring membrane-bound vesicles rather than L2. This is in line with previous observations that PML II is the only isoform binding to lipid droplets in the nucleus. We observed that the association of PML NBs with the HPV16 viral genome was transient and lost over time. We also found that nuclear Phospholipase C {beta}4 and {gamma}1 colocalize with PML II-L1 complexes in infected cells. Our data support a model in which PML II contributes to the establishment of early HPV16 infection by organizing host lipid regulatory factors at incoming viral transport vesicles.

cancer biology↗

bioRxiv2026

HPV16 Utilizes Phospholipase C(s) for its Genome Egress

Nuclear delivery of human papillomavirus (HPV) requires infected cells to undergo mitosis. Incoming HPV DNA is protected by a transport vesicle structure, which becomes unstable post-mitosis. We have previously shown that HPV genome egress takes on average 4 hours post-mitosis completion; thus, we propose that an enzymatic process is involved in degrading this transport vesicle to allow genome egress. Several phospholipase C (PLC) isoforms, including PLC{delta}3, PLC{zeta}1, and PLCL1, have been previously implicated in HPV infection in large-scale siRNA screens, and the parvovirus VP1 capsid protein has phospholipase enzymatic activity. Therefore, we hypothesized that cellular phospholipases residing in the nucleus mediate egress of the HPV genome from transport vesicles. We utilized siRNA-mediated knockdown and CRISPR/Cas9 knockout techniques to target specific PLC isoforms in both HeLa and HaCaT cell lines. We found that targeting PLC isoforms PLC{beta}1, {beta}4, {delta}3, {delta}4, and {gamma}1 significantly decreased HPV infection in both HeLa and HaCaT cells, as measured using a luciferase-based reporter assay. We also discovered a novel interaction between HPV16 minor protein L2 and PLCs {beta}1, {beta}4, {delta}1, {delta}3, and {delta}4. Furthermore, we observed that knockout of PLC{beta}4 and {delta}4 delayed egress of the HPV genome from nuclear membrane-bound vesicles after infection of HeLa cells. We propose that HPV utilizes phospholipase Cs to achieve genome egress from its protective vesicular structure after nuclear delivery.

microbiology↗

bioRxiv2022

Experimental support for human papillomavirus genome amplification early after infectious delivery

Even though replication and transcription of human papillomavirus type 16 (HPV16) has been intensively studied, little is known about immediate early events of the viral life cycle due to the lack of an efficient infection model allowing genetic dissection of viral factors. We employed the recently developed infection model (Bienkowska-Haba et al. PLOS Pathogen 2018) to investigate genome amplification and transcription immediately after infectious delivery of viral genome to nuclei of primary keratinocytes. Using EdU pulse labeling and highly sensitive fluorescence in situ hybridization, we observed that the HPV16 genome is replicated and amplified in an E1 and E2 dependent manner. Knockout of E1 resulted in failure of the viral genome to replicate and amplify. In contrast, knockout of the E8^E2 repressor led to increased viral genome copy number confirming previous reports. Genome copy control by E8^E2 was confirmed for differentiation-induced genome amplification. Lack of functional E1 had no effect on transcription from the early promoter, suggesting that viral genome replication is not required for p97 promoter activity. However, infection with an HPV16 mutant virus defective for E2 transcriptional function revealed a requirement of E2 for efficient transcription from the early promoter. In absence of the E8^E2 protein, early transcript levels are unaltered and even decreased when normalized to genome copy number. Surprisingly, lack of functional E8^E2 repressor did not affect E8^E2 transcript levels when normalized to genome copy number. These data suggest that the main function of E8^E2 in the viral life cycle is to control genome copy number. IMPORTANCEIt is being assumed that HPV utilizes three different modes of replication during its lifecycle: initial amplification during the establishment phase, genome maintenance and differentiation-induced amplification. However, HPV16 initial amplification was never formally proven due to a lack of an infection model. Using our recently established infection model (Bienkowska-Haba et al. PLOS Pathogen 2018), we demonstrate herein that viral genome is indeed amplified in an E1 and E2 dependent manner. Furthermore, we find that the main function of the viral repressor E8^E2 is to control viral genome copy number. We did not find evidence that it regulates its own promoter in a negative feedback loop. Our data also suggest that the E2 transactivator function is required for stimulation of early promoter activity, which has been debated in the literature. Overall, this report confirms the usefulness of the infection model for studying early events of the HPV lifecycle using mutational approaches.

microbiology↗

bioRxiv2022

Human Papillomavirus Genome Copy Number Is Maintained by S Phase Amplification, Leakage to the Cytosol during Mitosis and Lysosomal Degradation in G1 Phase

The current model for human papillomavirus (HPV) replication is comprised of three modes of replication. Following infectious delivery, the viral genome is amplified during the establishment phase to reach up to some hundred copies per cell. HPV genome copy number remains constant during the maintenance stage. Differentiation of infected cells induces HPV genome amplification. Using highly sensitive in situ hybridization (DNAscope) and freshly HPV16-infected as well as established HPV16-positive cell lines, we observed that viral genome is amplified in each S phase of undifferentiated keratinocytes cultured as monolayers. Nuclear viral genome copy number is reset to pre-S phase levels during mitosis. The majority of viral genome fails to tether to host chromosomes and is lost to the cytosol. Cytosolic viral genome gradually decreases during cell cycle progression. Loss of cytosolic genome is blocked in presence of NH4Cl or other drugs interfering with lysosomal acidification, suggesting the involvement of autophagy in viral genome degradation. These observations were also made with HPV31 cell lines obtained from patient samples. Cytosolic viral genome was not detected in UMSCC47 cells carrying integrated HPV16 DNA. Analyses of organotypic raft cultures derived from keratinocytes harboring episomal HPV16 revealed the presence of cytosolic viral genome as well. We conclude that HPV maintains viral genome copy number by balancing viral genome amplification during S phase with loss of viral genome that is lost to the cytosol during mitosis. It seems plausible that restrictions to viral genome tethering to mitotic chromosomes resets genome copy number in each cell cycle. IMPORTANCEHPV genome maintenance is currently being thought to be achieved by regulating expression and activity of viral replication factors E1 and E2. In addition, the E8^E2 repressor has been shown to be important for restricting genome copy number by competing with E1 and E2 for binding to the viral origin of replication and by recruitment of repressor complexes. Herein, we demonstrate that HPV viral genome is amplified in each S phase. Nuclear genome copy number is reset during mitosis by a failure of the majority of genomes to tether to mitotic chromosomes. Rather, they accumulate in the cytoplasm of freshly divided cells. Cytosolic viral DNA is quickly degraded in G1 in a lysosome dependent manner contributing to the genome copy reset. Our data imply that the mode of replication during establishment and maintenance is the same and further suggest that restrictions to genome tethering significantly contributes to viral genome maintenance.

microbiology↗