Novel replisome-associated proteins at cellular replication forks in EBV-transformed B lymphocytes

Abstract

Epstein-Barr virus (EBV) is an oncogenic herpesvirus and WHO class 1 carcinogen that resides in B lymphocytes of nearly all humans. While silent in most, EBV can cause endemic Burkitt lymphoma in children and post-transplant lymphoproliferative disorders/lymphomas in immunocompromised hosts. The pathogenesis of such lymphomas is multifactorial but to a large extent depends on EBV’s ability to aggressively drive cellular DNA replication and B cell proliferation despite cell-intrinsic barriers to replication. One such barrier is oncogenic replication stress which hinders the progression of DNA replication forks. To understand how EBV successfully overcomes replication stress, we examined cellular replication forks in EBV-transformed B cells using iPOND (isolation of Proteins on Nascent DNA)-mass spectrometry and identified several cellular proteins that had not previously been linked to DNA replication. Of eight candidate replisome-associated proteins that we validated at forks in EBV-transformed cells and Burkitt lymphoma-derived cells, three zinc finger proteins (ZFPs) were upregulated early in B cells newly-infected with EBV in culture as well as expressed at high levels in EBV-infected B blasts in the blood of immunocompromised transplant recipients. Expressed highly in S- and G2-phase cells, knockdown of each ZFP resulted in stalling of proliferating cells in the S-phase, cleavage of caspase 3, and cell death. These proteins, newly-identified at replication forks of EBV-transformed and Burkitt lymphoma cells therefore contribute to cell survival and cell cycle progression, and represent novel targets for intervention of EBV-lymphomas while simultaneously offering a window into how the replication machinery may be similarly modified in other cancers.

SARS-CoV-2 viroporin encoded by ORF3a triggers the NLRP3 inflammatory pathway

“…Flow cytometry was performed as previous described ( Xu et al, 2019a ). Briefly, HEK-293T and A549 cells were transfected with EV, FLAG-ORF3a, FLAG-ORF-E, or FLAG-ORF-8.…”

A heterochromatin inducing protein differentially recognizes self versus foreign genomes

“…With SZF1 being a DNA binding protein and its association, albeit low level, observed throughout the viral genome in sorted lytic cells ( S1 Fig ), we also asked if SZF1 might contribute to viral DNA replication originating from the lytic origins of replication. We therefore isolated nascent DNA using iPOND [ 29 ] from lytic-trigger exposed cells and found that while EA-D, as expected, was enriched at viral replication forks, SZF1 was not; consistent with pull-down of nascent viral DNA, the presence of PAA, a viral DNA polymerase inhibitor, demonstrated a reduction in DNA-bound EA-D ( Fig 8D ). Thus, on lytic genomes, SZF1 i) is enriched at the latent origin of replication, known to be silent during the lytic phase and ii) is not associated with actively replicating viral DNA.…”

“…1×10 8 HH514-16 cells were induced with 3mM NaB with or without phosphonoacetic acid (PAA) (200 μg/ml) for 36 hours prior to performing iPOND as described previously [ 29 ]. Briefly, cells were pulsed with 10 μM EdU for 15 min, spun down, cross-linked with 1% formaldehyde for 20 min, and quenched with 0.125 M glycine for 5 min.…”

Quantitative Proteomic Analysis of MARC-145 Cells Infected with a Mexican Porcine Reproductive and Respiratory Syndrome Virus Strain Using a Label-Free Based DIA approach

“…Zinc finger proteins (ZFP), the most widely occurring transcription actors which bind to nucleic acids and other molecules, play important roles in host immune response, and many of them are linked with carcinogenic processes and are involved in viral infections too. 79,80 Although there are no reports that directly involve ZNF428 with viral infections or processes related to cell division, several ZFPs have differentially expressed in various proteomic studies that demonstrate their participation in different mechanisms triggered by viral infections, such as endoplasmic reticulum stress and autophagy, 81 as well as alterations in DNA binding mediated by the inhibition of heat shock proteins 79 and forming part of replication forks, S-phase cell proliferation, and cell death. 80 All of these down-regulated molecules could be proteins affected by an inhibitory mechanism of cell division caused by PRRSV with the goal of ensuring its survival.…”