The dual role of the RETINOBLASTOMA-RELATED protein in the DNA damage response is spatio-temporally coordinated by the interaction with LXCXE-containing proteins.

Living organisms face threats to genome integrity caused by environmental challenges or metabolic errors in proliferating cells. To avoid the spread of mutations, cell division is temporarily arrested while repair mechanisms deal with DNA lesions. Afterwards, cells either resume division or respond to unsuccessful repair by withdrawing from the cell cycle and undergoing cell death. How the success rate of DNA repair connects to the execution of cell death remains incompletely known, particularly in plants. Here we provide evidence that the Arabidopsis thaliana RETINOBLASTOMA-RELATED1 (RBR) protein, shown to play structural and transcriptional functions in the DNA damage response (DDR), coordinates these processes in time by successive interactions through its B-pocket sub-domain. Upon DNA damage induction, RBR forms nuclear foci; but the N849F substitution in the B-pocket, which specifically disrupts binding to LXCXE motif-containing proteins, abolishes RBR focus formation and leads to growth arrest. After RBR focus formation, the stress-responsive gene NAC044 arrests cell division. As RBR is released from nuclear foci, it can be bound by the conserved LXCXE motif in NAC044. RBR-mediated cell survival is inhibited by the interaction with NAC044. Disruption of NAC044-RBR interaction impairs the cell death response but is less important for NAC044 mediated growth arrest. Noteworthy, unlike many RBR interactors, NAC044 binds to RBR independent of RBR phosphorylation. Our findings suggest that the availability of the RBR B-pocket to interact with LXCXE-containing proteins couples the structural DNA repair functions and the transcriptional functions of RBR in the cell death program.

SV40 Large T Antigen Is Not Responsible for the Loss of STING in 293T Cells but Can Inhibit cGAS-STING Interferon Induction

Several DNA viruses have evolved antagonists to inhibit the cyclic GMP–AMP synthase (cGAS)-stimulator of interferon genes (STING) DNA-sensing immune pathway. This includes DNA viral oncogenes that antagonize the cGAS-STING pathway by binding STING through the LxCxE motif. The 293T human cells are widely used in biology studies as they are highly transfectable. While parental 293 cells express high levels of STING, 293T cells lack STING and are unable to induce interferon antiviral responses to cytosolic DNA. Additionally, 293T cells express the SV40 polyomavirus large T antigen (LT) which enhances the replication of transfected DNA plasmids carrying the SV40 origin of replication. Since SV40 LT also encodes the LxCxE motif, the lack of STING expression in 293T cells is commonly assumed to be due to SV40 large T antigen. We find that SV40 LT does not alter exogenously expressed and endogenous levels of STING protein. We show that STING transcription is suppressed in 293T cells but is not driven by SV40. This study also revealed that SV40 LT does indeed inhibit cGAS-STING interferon induction, but through a mechanism distinct from other DNA virus oncogenes. Collectively, these results indicate that while SV40 LT can inhibit cGAS-STING interferon induction, it does so in an unanticipated manner.

Complete genome and phylogenetic analysis of bovine papillomavirus type 15 in Southern Xinjiang dairy cow

In this study, the complete genome sequence of bovine papillomavirus (BPV) type 15 (BPV Aks-02), a novel putative BPV type from a skin sample of a cow in southern Xinjiang, China was determined by collecting cutaneous neoplastic lesion, followed by DNA extraction and amplicon sequencing. The complete genome consisted of 7189 base pairs (G+C content of 42.50%) that encoded five early (E8, E7, E1, E2, E4) and two late (L1 and L2) genes. The E7 protein contained a consensus CX2CX29CX2C zinc-binding domain and an LxCxE motif. The nucleotide sequence of the L1 open reading frame (ORF) was related mostly (99%) to the L1 ORF of putative type BAPV-3 reference strain from GenBank. Phylogenetic analysis and sequence similarities based on the L1 ORF suggest that BPV type (BPV Aks-02) clustered with members of genus Xipapillomavirus as BPV15, and closely related to Xipapillomavirus 1.

Complete genome and phylogenetic position of bovine papillomavirus type 7

Six bovine papillomavirus (BPV) types and 16 putative BPV types have been reported previously. Here, the complete genome sequence of BAPV6, a novel putative BPV type isolated from cattle in Japan, was determined by using multiple-primed rolling-circle amplification. The genome consisted of 7412 bp (G+C content of 46 mol%) that encoded five early (E1, E2, E4, E6 and E7) and two late (L1 and L2) genes, but did not encode the E5 gene. The E6 protein contained a non-consensus CxxC(x)33CxxC and a consensus CxxC(x)29CxxC zinc-binding domain, and the E7 protein lacked the LxCxE motif. The nucleotide sequence of the L1 open reading frame (ORF) was related most closely (57–58 %) to the L1 ORF of member(s) of the genera Betapapillomavirus, Gammapapillomavirus and Pipapillomavirus. Phylogenetic analysis based on the complete L1 ORF suggests that BAPV6 should be classified in a novel genus in the family Papillomaviridae as BPV-7.

The Direct Association of GATA-1 to Rb Is Necessary for Definitive Erythropoiesis.

GATA-1, the founding member of GATA family, is a master gene for erythropoiesis. It binds to the consensus sequence WGATAR and is involved in the activation of most of the erythroid specific genes. GATA-1 can also repress the transcription of genes coding for proteins involved in the control of cell proliferation. Here we describe a novel function of GATA-1 i.e. the control of the cell proliferation through a direct association to the retinoblastoma protein (pRb). This association required the canonical LXCXE motif localized between amino acids 81 and 85 of GATA-1. This sequence is conserved during evolution, is not found in the other members of the GATA family and is absent in the short form of GATA-1 expressed in the acute megakaryoblastic leukemia of Down syndrome. The inhibition of cellular proliferation induced by GATA-1 over-expression was inefficient in pRb negative cell lines or when pRb was knocked down by specific shRNA and, using a GATA-1 protein mutated on the LXCXE motif (GATA-1Rb-), we showed that (i) the LXCXE motif was necessary for GATA-1/pRb association, (ii) the over-expression of GATA-1Rb- did not inhibit cellular proliferation whereas GATA-1 over-expression did, and (iii) the expression of GATA-1Rb- in a GATA-1 deficient erythroid cell line decreased the number of benzidine positive cells and increased the immature erythroid fraction. Finally, endogenous level expression of GATA-1Rb- with GATA-1 HRD vector in the GATA-1.05 genotype mice resulted in a partial rescue of GATA-1.05 embryos from the early lethality at E11.5, but the embryos then exhibited severe anemia and died by E15.5. Analysis of the E12.5 fetal liver of these GATA-1Rb- rescued mice showed an increased number of immature erythroid cells and a decreased number of mature erythroid cells. These results indicate that the GATA-1/pRB interaction is necessary for erythropoiesis enlighting the mechanism of the autonomous defect of erythropoiesis observed in the Rb−/− mice.

Destabilization of the Retinoblastoma Tumor Suppressor by Human Papillomavirus Type 16 E7 Is Not Sufficient To Overcome Cell Cycle Arrest in Human Keratinocytes

ABSTRACT The E7 oncoprotein of human papillomavirus type 16 promotes cell proliferation in the presence of antiproliferative signals. Mutagenesis of E7 has revealed that this activity requires three regions, conserved regions 1 and 2 and a C-terminal zinc finger. Binding to the retinoblastoma tumor repressor (Rb) through an LxCxE motif in conserved region 2 is necessary, but not sufficient, for E7 to induce proliferation. We tested the hypothesis that binding to Rb is not sufficient because conserved region 1 and/or the C terminus are required for E7 to functionally inactivate Rb and thus induce proliferation. One mechanism proposed for how E7 inactivates Rb is by blocking Rb-E2F binding. Either conserved region 1 or the C terminus was necessary, in combination with the LxCxE motif, for E7 to block Rb-E2F binding in vitro. While all full-length E7 proteins with mutations outside of the LxCxE motif inhibited Rb-E2F binding, some failed to abrogate cell cycle arrest, demonstrating that blocking Rb-E2F binding is not sufficient for abrogating antiproliferative signals. Another mechanism proposed for how E7 inactivates Rb is by promoting the destabilization of Rb protein. Mutations in conserved region 1 or the LxCxE motif prevented E7 from reducing the half-life of Rb. Though no specific C-terminal residues of E7 were essential for destabilizing Rb, a novel class of mutations that uncouple the destabilization of Rb from the deregulation of keratinocyte proliferation was discovered. Destabilization of Rb correlated with the abrogation of Rb-induced quiescence but was not sufficient for overriding DNA damage-induced cell cycle arrest or for increasing keratinocyte life span. Finally, the same regions of E7 required for destabilizing Rb were required for reducing p107 and p130 levels. Together, these results suggest that inactivation of all three Rb family members is not sufficient to deregulate keratinocyte cell cycle control.

Loss of p19ARF Eliminates the Requirement for the pRB-Binding Motif in Simian Virus 40 Large T Antigen-Mediated Transformation

ABSTRACT At least three domains of simian virus 40 large T antigen (TAg) participate in cellular transformation. The LXCXE motif of TAg binds to all members of the retinoblastoma protein (pRB) family of tumor suppressors. The N-terminal 70 residues of TAg have significant homology to the J domain of Hsp40/DnaJ and cooperate with the LXCXE motif to inactivate the pRB family. A bipartite C-terminal domain of TAg binds to p53 and thereby disrupts the ability of p53 to act as a sequence-specific transcription factor. The contribution of these three domains of TAg to cellular transformation was evaluated in cells that contained inactivating mutations in the pRB and p53 pathways. Cells that stably expressed wild-type or selected mutant forms of TAg were generated in mouse embryo fibroblasts (MEFs) containing homozygous deletions in the RB, INK4a, and ARFloci. It was determined that the J domain, the LXCXE motif, and the p53-binding domain of TAg were required for full transformation of wild-type and RB −/− MEFs. In contrast,INK4a −/− MEFs that lacked expression of p16 INK4a and p19 ARF andARF −/− MEFs that lacked p19 ARF but expressed p16 INK4a acquired anchorage-independent growth when expressing wild-type TAg or mutant derivatives that disrupted either the pRB-binding or p53-binding domain. The expression and function of the pRB family members were not overly disrupted inARF −/− MEFs expressing LXCXE mutants of TAg. These results suggest that inactivating mutations of p19 ARF can relieve the requirement for the LXCXE motif in TAg-mediated transformation and that TAg may have additional functions in transformation.