Interaction of 2A proteinase of human rhinovirus genetic group A with eIF4E is required for eIF4G cleavage during infection

In enteroviruses, the inhibition of protein synthesis from capped host cell mRNA is catalyzed by the virally encoded 2A proteinase (2Apro), which cleaves eukaryotic initiation factors (eIF) 4GI and 4GII. Despite much investigation, the exact mechanism of 2Apro cleavage remains however unclear. Here, we identify the domains responsible for the eIF4E/HRV2 2Apro interaction using molecular modelling and describe mutations that impair this interaction and delay in vitro cleavage of eIF4G isoforms. Furthermore, we produced HRV1A viruses bearing the mutation L17R, Y32A or Y86A in the 2Apro sequence. All three viruses showed reduced yield and were appreciably delayed during infection in eIF4GI cleavage. Thus, we propose for genetic group A HRVs that the eIF4E/2Apro interaction is essential for successful viral replication. In contrast, HRV4 2Apro and coxsackievirus B4 2Apro failed to form complexes with eIF4E, suggesting that the mechanism of eIF4G isoform cleavage in these and related viruses is different.(VLID)485648

Engagement of SIRP alpha Inhibits Growth and Induces Programmed Cell Death in Acute Myeloid Leukemia Cells

<p>Background: Recent studies show the importance of interactions between CD47 expressed on acute myeloid leukemia (AML) cells and the inhibitory immunoreceptor, signal regulatory protein-alpha (SIRP alpha) on macrophages. Although AML cells express SIRP alpha, its function has not been investigated in these cells. In this study we aimed to determine the role of the SIRP alpha in acute myeloid leukemia.</p><p>Design and Methods: We analyzed the expression of SIRP alpha, both on mRNA and protein level in AML patients and we further investigated whether the expression of SIRP alpha on two low SIRP alpha expressing AML cell lines could be upregulated upon differentiation of the cells. We determined the effect of chimeric SIRP alpha expression on tumor cell growth and programmed cell death by its triggering with an agonistic antibody in these cells. Moreover, we examined the efficacy of agonistic antibody in combination with established antileukemic drugs.</p><p>Results: By microarray analysis of an extensive cohort of primary AML samples, we demonstrated that SIRP alpha is differentially expressed in AML subgroups and its expression level is dependent on differentiation stage, with high levels in FAB M4/M5 AML and low levels in FAB M0-M3. Interestingly, AML patients with high SIRP alpha expression had a poor prognosis. Our results also showed that SIRP alpha is upregulated upon differentiation of NB4 and Kasumi cells. In addition, triggering of SIRP alpha with an agonistic antibody in the cells stably expressing chimeric SIRP alpha, led to inhibition of growth and induction of programmed cell death. Finally, the SIRP alpha-derived signaling synergized with the activity of established antileukemic drugs.</p><p>Conclusions: Our data indicate that triggering of SIRP alpha has antileukemic effect and may function as a potential therapeutic target in AML.</p>

A novel non-canonical PIP-box mediates PARG interaction with PCNA

Poly(ADP-ribose) glycohydrolase (PARG) regulates cellular poly(ADP-ribose) (PAR) levels by rapidly cleaving glycosidic bonds between ADP-ribose units. PARG interacts with proliferating cell nuclear antigen (PCNA) and is strongly recruited to DNA damage sites in a PAR- and PCNA-dependent fashion. Here we identified PARG acetylation site K409 that is essential for its interaction with PCNA, its localization within replication foci and its recruitment to DNA damage sites. We found K409 to be part of a non-canonical PIP-box within the PARG disordered regulatory region. The previously identified putative N-terminal PIP-box does not bind PCNA directly but contributes to PARG localization within replication foci. X-ray structure and MD simulations reveal that the PARG non-canonical PIP-box binds PCNA in a manner similar to other canonical PIP-boxes and may represent a new type of PIP-box. While the binding of previously described PIP-boxes is based on hydrophobic interactions, PARG PIP-box binds PCNA via both stabilizing hydrophobic and fine-tuning electrostatic interactions. Our data explain the mechanism of PARG–PCNA interaction through a new PARG PIP-box that exhibits non-canonical sequence properties but a canonical mode of PCNA binding.© The Author(s) 201