Identification and characterization of Risp

The aim of this study was to characterize the role of Rev-interacting cellular proteins in controlling the function of Rev in the host cell. The HIV-1 protein Rev plays an essential role in the temporal regulation of the virus gene expression by stimulating the expression of viral structural proteins. Rev enhances the nucleocytoplasmic transport and the translation of unspliced and single spliced viral mRNAs by binding with high affinity to a specific target element on the HIV-RNA. It was assumed that interaction with cellular factors is essential for Rev function. At the onset of this study, only a few potential cofactors were known with no clearly defined functional relevance. So we decided to search for new Rev-interacting factors using the yeast two-hybrid system. In this work a new Rev-interacting protein has been identified, by screening a Jurkat T cell cDNA library. The protein was termed Risp (Rev-interacting shuttle protein), because it shuttles between the nuclear and the cytoplasmic compartments. The Risp gene is widely expressed in human cells and conserved among various species, most probably as part of a larger gene. High amino acid homology (99%) with the C-terminal part of a large brain cDNA clone for KIAA0592 protein has been found, whereas no obvious homology to proteins with known function was observed. However, a weak and partial similarity appeared with several RNA-/DNA-binding and shuttle proteins. This might indicate that the Risp protein - or the larger protein containing it - could be a member of a new family of nucleocytoplasmic shuttle proteins with RNA-/DNA-binding function. Next, the intracellular localization and shuttling of Risp was investigated. In HeLa cells Risp- GFP localized in both nuclear and cytoplasmic compartments, but clearly accumulated in the cytoplasm, indicating the presence of a strong nuclear export signal (NES). The identification of a NES sequence was confirmed by deletion analysis of Risp and by nuclear microinjection of BSA-fusion proteins conjugated to peptides from the C-terminal part of Risp. Treatment with leptomycin B, a drug which has been shown to specifically block Crm1 (exportin) mediated export, resulted in nuclear accumulation of Risp-GFP, showing that the nuclear export of Risp, like that of Rev, is Crm1-dependent. Using bioinformatic tools able to detect weak homologies with high specificity, sequence comparisons between Risp and all currently known Rev interacting factors were performed. This analysis for the first time revealed a common motif shared between Rev and Rev-interacting cellular factors, termed RIP. The region of Risp harboring the RIP motif was neither essential nor sufficient for the Rev-binding in the yeast two hybrid system, suggesting no direct correlation between RIP and the Rev-binding ability. Preliminary experiments suggested, that Risp, as a Rev-interacting protein, is able to inhibit Revtrans- activation, while Risp does not interfere with Tat in a Tat-trans-activation assay. The overexpression of Risp-GFP reduced the production of the Rev-dependent structural viral protein p24gag up to 70%. In addition a previously unrecognized sequence motif in the activation domain of Rev with intrinsic nuclear import activity was found and tested in transfection and microinjection assays. This motif (“PPXXR”) is conserved in various RNA-binding proteins and was proposed to mediate nuclear translocation of the cellular functional homologue of HIV-1 Rev Sam68

Multipass automata and group word problems

We introduce the notion of multipass automata as a generalization of pushdown automata and study the classes of languages accepted by such machines. The class of languages accepted by deterministic multipass automata is exactly the Boolean closure of the class of deterministic context-free languages while the class of languages accepted by nondeterministic multipass automata is exactly the class of poly-context-free languages, that is, languages which are the intersection of finitely many context-free languages. We illustrate the use of these automata by studying groups whose word problems are in the above classes

Topological properties of cellular automata on trees

We prove that there do not exist positively expansive cellular automata defined on the full k-ary tree shift (for k>=2). Moreover, we investigate some topological properties of these automata and their relationships, namely permutivity, surjectivity, preinjectivity, right-closingness and openness.Comment: In Proceedings AUTOMATA&JAC 2012, arXiv:1208.249

Selected amino acid mutations in HIV-1 B subtype gp41 are Associated with Specific gp120V3 signatures in the regulation of Co-Receptor usage

<p>Abstract</p> <p>Background</p> <p>The third variable loop (V3) of the HIV-1 gp120 surface protein is a major determinant of cellular co-receptor binding. However, HIV-1 can also modulate its tropism through other regions in gp120, such as V1, V2 and C4 regions, as well as in the gp41 protein. Moreover, specific changes in gp41 are likely to be responsible for of damage in gp120-CCR5 interactions, resulting in potential resistance to CCR5 inhibitors.</p> <p>In order to genetically characterize the two envelope viral proteins in terms of co-receptor usage, we have analyzed 526 full-length <it>env </it>sequences derived from HIV-1 subtype-B infected individuals, from our and public (Los Alamos) databases. The co-receptor usage was predicted by the analysis of V3 sequences using Geno2Pheno (G2P) algorithm. The binomial correlation phi coefficient was used to assess covariation among gp120_V3and gp41 mutations; subsequently the average linkage hierarchical agglomerative clustering was performed.</p> <p>Results</p> <p>According to G2P false positive rate (FPR) values, among 526 env-sequences analyzed, we further characterized 196 sequences: 105 with FPR <5% and 91 with FPR >70%, for X4-using and R5-using viruses, respectively.</p> <p>Beyond the classical signatures at 11/25 V3 positions (S11S and E25D, R5-tropic viruses; S11KR and E25KRQ, X4-tropic viruses), other specific V3 and gp41 mutations were found statistically associated with the co-receptor usage. Almost all of these specific gp41 positions are exposed on the surface of the glycoprotein. By the covariation analysis, we found several statistically significant associations between V3 and gp41 mutations, especially in the context of CXCR4 viruses. The topology of the dendrogram showed the existence of a cluster associated with R5-usage involving E25D_V3, S11S_V3, T22A_V3, S129DQ_gp41and A96N_gp41signatures (bootstrap = 0.88). Conversely, a large cluster was found associated with X4-usage involving T8I_V3, S11KR_V3, F20IVY_V3, G24EKR_V3, E25KR_V3, Q32KR_V3, A30T_gp41, A189S_gp41, N195K_gp41and L210P_gp41mutations (bootstrap = 0.84).</p> <p>Conclusions</p> <p>Our results show that gp120_V3and several specific amino acid changes in gp41 are associated together with CXCR4 and/or CCR5 usage. These findings implement previous observations that determinants of tropism may reside outside the V3-loop, even in the gp41. Further studies will be needed to confirm the degree to which these gp41 mutations contribute directly to co-receptor use.</p

Groups, Graphs, Languages, Automata, Games and Second-order Monadic Logic

In this paper we survey some surprising connections between group theory, the theory of automata and formal languages, the theory of ends, infinite games of perfect information, and monadic second-order logic