The gammaretroviral p12 protein has multiple domains that function during the early stages of replication.

BACKGROUND: The Moloney murine leukaemia virus (Mo-MLV) gag gene encodes three main structural proteins, matrix, capsid and nucleocapsid and a protein called p12. In addition to its role during the late stages of infection, p12 has an essential, but undefined, function during early post-entry events. As these stages of retroviral infection remain poorly understood, we set out to investigate the function of p12. RESULTS: Examination of the infectivity of Mo-MLV virus-like particles containing a mixture of wild type and mutant p12 revealed that the N- and C-terminal regions of p12 are sequentially acting domains, both required for p12 function, and that the N-terminal activity precedes the C-terminal activity in the viral life cycle. By creating a panel of p12 mutants in other gammaretroviruses, we showed that these domains are conserved in this retroviral genus. We also undertook a detailed mutational analysis of each domain, identifying residues essential for function. These data show that different regions of the N-terminal domain are necessary for infectivity in different gammaretroviruses, in stark contrast to the C-terminal domain where the same region is essential for all viruses. Moreover, chimeras between the p12 proteins of Mo-MLV and gibbon ape leukaemia virus revealed that the C-terminal domains are interchangeable whereas the N-terminal domains are not. Finally, we identified potential functions for each domain. We observed that particles with defects in the N-terminus of p12 were unable to abrogate restriction factors, implying that their cores were impaired. We further showed that defects in the C-terminal domain of p12 could be overcome by introducing a chromatin binding motif into the protein. CONCLUSIONS: Based on these data, we propose a model for p12 function where the N-terminus of p12 interacts with, and stabilizes, the viral core, allowing the C-terminus of p12 to tether the preintegration complex to host chromatin during mitosis, facilitating integration

Replication gap suppression depends on the double-strand DNA binding activity of BRCA2

Replication stress (RS) is a major source of genomic instability and is intrinsic to cancer cells. RS is also the consequence of chemotherapeutic drugs for treating cancer. However, adaptation to RS is also a mechanism of resistance to chemotherapy. BRCA2 deficiency results in replication stress in human cells. BRCA2 protein’s main functions include DNA repair by homologous recombination (HR) both at induced DNA double-strand breaks (DSB) and spontaneous replicative lesions. At stalled replication forks, BRCA2 protects the DNA from aberrant nucleolytic degradation and is thought to limit the appearance of ssDNA gaps by arresting replication and via post-replicative HR. However, whether and how BRCA2 acts to limit the formation of ssDNA gaps or mediate their repair, remains ill-defined. Here, we use breast cancer variants affecting different domains of BRCA2 to shed light on this function. We demonstrate that the N-terminal DNA binding domain (NTD), and specifically, its dsDNA binding activity, is required to prevent and repair/fill-in ssDNA gaps upon nucleotide depletion but not to limit PARPi-induced ssDNA gaps. Thus, these findings suggest that nucleotide depletion and PARPi trigger gaps via distinct mechanisms and that the NTD of BRCA2 prevents nucleotide depletion-induced ssDNA gaps.This research was funded by grants from the Agence National de Recherche (ANR-17-CE12-0016), Institut National du Cancer (INCa-DGOS_8706), and Agencia Española de Investigacion (MCIN/AEI) (PID2020-115977RB-I00) to A.C. and Jeunes Chercheuses from the Agence National de la Recherche (REPLIBLOCK ANR-17-CE12-0034-01) to C.R. A predoctoral Fellowship from Fondation ARC pour la recherche sur le cancer and French Ministry of Education supported D.V. A predoctoral Fellowship from Fondation pour la Recherche Medicale (FRM) supported I.D., A.M., and C.v.N. L.A-A. was funded by an FPI Fellowship from Agencia Española de Investigacion (MCIN/AEI) (PID2020-115977RB-I00). R.C. was funded by a PSL (Paris Science et Lettres) predoctoral Fellowship and R.L. was funded by Fondation ARC pour la recherche sur le cancer

Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome.

BACKGROUND: Detection of a retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), has recently been reported in 67% of patients with chronic fatigue syndrome. We have studied a total of 170 samples from chronic fatigue syndrome patients from two UK cohorts and 395 controls for evidence of XMRV infection by looking either for the presence of viral nucleic acids using quantitative PCR (limit of detection <16 viral copies) or for the presence of serological responses using a virus neutralisation assay. RESULTS: We have not identified XMRV DNA in any samples by PCR (0/299). Some serum samples showed XMRV neutralising activity (26/565) but only one of these positive sera came from a CFS patient. Most of the positive sera were also able to neutralise MLV particles pseudotyped with envelope proteins from other viruses, including vesicular stomatitis virus, indicating significant cross-reactivity in serological responses. Four positive samples were specific for XMRV. CONCLUSIONS: No association between XMRV infection and CFS was observed in the samples tested, either by PCR or serological methodologies. The non-specific neutralisation observed in multiple serum samples suggests that it is unlikely that these responses were elicited by XMRV and highlights the danger of over-estimating XMRV frequency based on serological assays. In spite of this, we believe that the detection of neutralising activity that did not inhibit VSV-G pseudotyped MLV in at least four human serum samples indicates that XMRV infection may occur in the general population, although with currently uncertain outcomes

Deubiquitinase USP8 targets ESCRT-III to promote incomplete cell division

International audienceIn many vertebrate and invertebrate organisms, gametes develop within groups of interconnected cells called germline cysts formed by several rounds of incomplete divisions. We found that loss of the deubiquitinase USP8 gene in Drosophila can transform incomplete divisions of germline cells into complete divisions. Conversely, overexpression of USP8 in germline stem cells is sufficient for the reverse transformation from complete to incomplete cytokinesis. The ESCRT-III proteins CHMP2B and Shrub/CHMP4 are targets of USP8 deubiquitinating activity. In Usp8 mutant sister cells, ectopic recruitment of ESCRT proteins at intercellular bridges causes cysts to break apart. A Shrub/CHMP4 variant that cannot be ubiquitinated does not localize at abscission bridges and cannot complete abscission. Our results uncover ubiquitination of ESCRT-III as a major switch between two types of cell division

The nuclear pore primes recombination-dependent DNA synthesis at arrested forks by promoting SUMO removal

International audienceNuclear Pore complexes (NPCs) act as docking sites to anchor particular DNA lesions facilitating DNA repair by elusive mechanisms. Using replication fork barriers in fission yeast, we report that relocation of arrested forks to NPCs occurred after Rad51 loading and its enzymatic activity. The E3 SUMO ligase Pli1 acts at arrested forks to safeguard integrity of nascent strands and generates poly-SUMOylation which promote relocation to NPCs but impede the resumption of DNA synthesis by homologous recombination (HR). Anchorage to NPCs allows SUMO removal by the SENP SUMO protease Ulp1 and the proteasome, promoting timely resumption of DNA synthesis. Preventing Pli1-mediated SUMO chains was sufficient to bypass the need for anchorage to NPCs and the inhibitory effect of poly-SUMOylation on HR-mediated DNA synthesis. Our work establishes a novel spatial control of Recombination-Dependent Replication (RDR) at a unique sequence that is distinct from mechanisms engaged at collapsed-forks and breaks within repeated sequences

RNA:DNA hybrids from Okazaki fragment contribute to establish the Ku-mediated barrier to replication fork degradation

International audienceNon-homologous end-joining (NHEJ) factors act in replication fork protection, restart and repair. Here, we identified a mechanism related to RNA:DNA hybrids to establish the NHEJ factor Ku-mediated barrier to nascent strand degradation, in fission yeast. RNase H activities promote nascent strand degradation and replication restart, with a prominent role of RNase H2 in processing RNA:DNA hybrids to overcome the Ku-barrier to nascent strand degradation. RNase H2 co-operates with the MRN-Ctp1 axis to sustain cell resistance to replication stress, in a Ku-dependent manner. Mechanistically, the need of RNaseH2 in nascent strand degradation requires the primase activity that allows establishing the Ku-barrier to Exo1 whereas impairing Okazaki fragment maturation reinforces the Ku-barrier. Finally, replication stress induces Ku foci in a primase-dependent manner and favors Ku binding to RNA:DNA hybrids. We propose a function for the RNA:DNA hybrid originating from Okazaki fragment in controlling the Ku-barrier specifying nuclease requirement to engage fork resection

Proper chromosome alignment depends on BRCA2 phosphorylation by PLK1

International audienceThe BRCA2 tumor suppressor protein is involved in the maintenance of genome integrity through its role in homologous recombination. In mitosis, BRCA2 is phosphorylated by Polo-like kinase 1 (PLK1). Here we describe how this phosphorylation contributes to the control of mitosis. We identify a conserved phosphorylation site at T207 of BRCA2 that constitutes a bona fide docking site for PLK1 and is phosphorylated in mitotic cells. We show that BRCA2 bound to PLK1 forms a complex with the phosphatase PP2A and phosphorylated-BUBR1. Reducing BRCA2 binding to PLK1, as observed in BRCA2 breast cancer variants S206C and T207A, alters the tetrameric complex resulting in unstable kinetochore-microtubule interactions, misaligned chromosomes, faulty chromosome segregation and aneuploidy. We thus reveal a role of BRCA2 in the alignment of chromosomes, distinct from its DNA repair function, with important consequences on chromosome stability. These findings may explain in part the aneuploidy observed in BRCA2-mutated tumors

<i>Tropheryma whipplei</i>, the Agent of Whipple's Disease, Affects the Early to Late Phagosome Transition and Survives in a Rab5- and Rab7-Positive Compartment

<div><p><i>Tropheryma whipplei</i>, the agent of Whipple's disease, inhibits phago-lysosome biogenesis to create a suitable niche for its survival and replication in macrophages. To understand the mechanism by which it subverts phagosome maturation, we used biochemical and cell biological approaches to purify and characterise the intracellular compartment where <i>Tropheryma whipplei</i> resides using mouse bone-marrow-derived macrophages. We showed that in addition to Lamp-1, the <i>Tropheryma whipplei</i> phagosome is positive for Rab5 and Rab7, two GTPases required for the early to late phagosome transition. Unlike other pathogens, inhibition of PI(3)P production was not the mechanism for Rab5 stabilisation at the phagosome. Overexpression of the inactive, GDP-bound form of Rab5 bypassed the pathogen-induced blockade of phago-lysosome biogenesis. This suggests that <i>Tropheryma whipplei</i> blocks the switch from Rab5 to Rab7 by acting on the Rab5 GTPase cycle. A bio-informatic analysis of the <i>Tropheryma whipplei</i> genome revealed a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) homologous with the GAPDH of <i>Listeria monocytogenes</i>, and this may be the bacterial protein responsible for blocking Rab5 activity. To our knowledge, <i>Tropheryma whipplei</i> is the first pathogen described to induce a “chimeric” phagosome stably expressing both Rab5 and Rab7, suggesting a novel and specific mechanism for subverting phagosome maturation.</p></div

<i>T. whipplei</i> survives and replicates in a Lamp-1- but not cathepsin D-positive compartment within BMDMs.

<p>(<b>A</b>) BMDMs were incubated with <i>T. whipplei</i> (bacterium-to-cell ratio of 50∶1) for 4 hours (day 0). Cells were then washed to remove free bacteria and incubated for additional periods (days). The copy number of bacterial DNA was determined by qRT-PCR. The results are expressed as the mean ± SD from 3 experiments (*<i>p</i><0.05). (<b>B–D</b>) The colocalisation of <i>T whipplei</i> with either (<b>B</b>) Lamp-1 or (<b>C</b>) cathepsin D was analysed in BMDMs by immunofluorescence and confocal microscopy. The percentage of <i>T whipplei</i> colocalising with either Lamp-1 or cathepsin D was quantified over the time (<b>D</b>). The results are expressed as the mean ± SD from 3 experiments (*<i>p</i><0.05). The scale bars indicate 5 µm.</p