Phosphorylation of the RSRSP stretch is critical for splicing regulation by RNA-Binding Motif Protein 20 (RBM20) through nuclear localization

RBM20 is a major regulator of heart-specific alternative pre-mRNA splicing of TTN encoding a giant sarcomeric protein titin. Mutation in RBM20 is linked to autosomal-dominant familial dilated cardiomyopathy (DCM), yet most of the RBM20 missense mutations in familial and sporadic cases were mapped to an RSRSP stretch in an arginine/serine-rich region of which function remains unknown. In the present study, we identified an R634W missense mutation within the stretch and a G1031X nonsense mutation in cohorts of DCM patients. We demonstrate that the two serine residues in the RSRSP stretch are constitutively phosphorylated and mutations in the stretch disturb nuclear localization of RBM20. Rbm20 S637A knock-in mouse mimicking an S635A mutation reported in a familial case showed a remarkable effect on titin isoform expression like in a patient carrying the mutation. These results revealed the function of the RSRSP stretch as a critical part of a nuclear localization signal and offer the Rbm20 S637A mouse as a good model for in vivo study

Protection of Macaques with Diverse MHC Genotypes against a Heterologous SIV by Vaccination with a Deglycosylated Live-Attenuated SIV

HIV vaccine development has been hampered by issues such as undefined correlates of protection and extensive diversity of HIV. We addressed these issues using a previously established SIV-macaque model in which SIV mutants with deletions of multiple gp120 N-glycans function as potent live attenuated vaccines to induce near-sterile immunity against the parental pathogenic SIVmac239. In this study, we investigated the protective efficacy of these mutants against a highly pathogenic heterologous SIVsmE543-3 delivered intravenously to rhesus macaques with diverse MHC genotypes. All 11 vaccinated macaques contained the acute-phase infection with blood viral loads below the level of detection between 4 and 10 weeks postchallenge (pc), following a transient but marginal peak of viral replication at 2 weeks in only half of the challenged animals. In the chronic phase, seven vaccinees contained viral replication for over 80 weeks pc, while four did not. Neutralizing antibodies against challenge virus were not detected. Although overall levels of SIV specific T cell responses did not correlate with containment of acute and chronic viral replication, a critical role of cellular responses in the containment of viral replication was suggested. Emergence of viruses with altered fitness due to recombination between the vaccine and challenge viruses and increased gp120 glycosylation was linked to the failure to control SIV. These results demonstrate the induction of effective protective immune responses in a significant number of animals against heterologous virus by infection with deglycosylated attenuated SIV mutants in macaques with highly diverse MHC background. These findings suggest that broad HIV cross clade protection is possible, even in hosts with diverse genetic backgrounds. In summary, results of this study indicate that deglycosylated live-attenuated vaccines may provide a platform for the elucidation of correlates of protection needed for a successful HIV vaccine against diverse isolates

Laz İsmail, Nazım abimi öldürmek istedi

Taha Toros Arşivi, Dosya Adı: Nazım Hikmetİstanbul Kalkınma Ajansı (TR10/14/YEN/0033) İstanbul Development Agency (TR10/14/YEN/0033

Gag_206–216, Gag_241–249, and Gag_367–381 epitope-specific CD8⁺ T-cell responses in SIV controllers.

<p>(A) Frequencies of CD8⁺ T cells specific for SIV Gag_206–216, Gag_241–249, and Gag_367–381 epitopes in Group M (upper panels) and Group N (lower) at 4 months (4M), 1 year (1Y), and 2 years (2Y) post-infection. (B) Comparisons of the sum of Gag_206–216-, Gag_241–249-, and Gag_367–381-specific CD8⁺ T-cell frequencies at 4M, 1Y, and 2Y between Groups M and N. No significant difference was observed between the groups.</p

Dominant non-synonymous mutations in proviral <i>gag</i> in SIV controllers.

<p>Amino acid substitutions around SIV Gag_206–216, Gag_241–249, and Gag_367–381 epitopes and in other Gag regions approximately 2 months (2M, top), 1 year (1Y, middle), and 2 years (2Y, bottom) after SIVmac239 challenge are shown. Most of the proviral gag fragments were amplified from CD4⁺ T cells isolated from PBMCs, while those at 2 years in macaques R06-037, R05-005, R07-001, and R07-006 were from cultured CD4⁺ T cells due to limitation of available cell numbers. Mutant sequences shown were completely dominant (i.e., wild-type sequences were undetectable at the residues showing mutant sequences) except for the L216S mutation (the ratio of wild type/mutant: 2/5) in macaque R03-018 at 1 year post-infection. No subdominant mutation was detected.</p

Nef_9–19, Nef_89–97, Nef_193–203, and Vif_114–124 epitope-specific CD8⁺ T-cell responses in SIV controllers.

<p>(A) Frequencies of CD8⁺ T cells specific for SIV Nef_9–19, Nef_89–97, Nef_193–203, and Vif_114–124 epitopes in Group M (upper panels) and Group N (lower) at 4 months (4M), 1 year (1Y), and 2 years (2Y) post-infection. (B) Comparisons of the sum of Nef_9–19-, Nef_89–97-, Nef_193–203-, and Vif_114–124-specific CD8⁺ T-cell frequencies at 4M, 1Y, and 2Y between Groups M and N. The sum of CD8⁺ T-cell frequencies specific for these epitopes in Group M was significantly higher compared to Group N at 2Y post-infection (p = 0.0190 by Mann-Whitney U-test).</p

Dominant non-synonymous mutations in proviral <i>vif</i> and <i>nef</i> in SIV controllers.

<p>In the upper panel, amino acid substitutions around SIV Vif_114–124 epitope and in other Vif regions approximately 2 years after SIVmac239 challenge are shown. In the lower panel, amino acid substitutions around SIV Nef_9–19, Nef_89–97, and Nef_193–203 epitopes and in other Nef regions approximately 2 years after SIVmac239 challenge are shown. Sequences of <i>vif</i> in macaques R03-018 and R07-008 and <i>nef</i> in macaques R07-008 were not determined because these cDNA fragments could not be amplified. Macaques R07-001 and R07-003 had multiple G-to-A mutations in <i>nef</i> (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005247#ppat.1005247.s001" target="_blank">S1 Fig</a>). Mutant sequences shown were completely dominant except for <i>vif</i> P138L (the ratio of wild type/mutant: 1/2) in R05-005, <i>vif</i> Q162R (1/1) in R07-006, <i>nef</i> P012T (1/10) in R06-037, and <i>nef</i> S013P (3/10), I090T (1/5), D096N (1/5), and E191K (1/5) in R07-002. In addition, subdominant <i>nef</i> mutations resulting in P012S (5/2) and G044E (5/2) were detected in macaque R07-002, while the wild-type sequences were dominant at these positions.</p

Virological and immunological analyses in macaque R09-009 following CD8⁺ cell depletion.

<p>(A) Changes in peripheral CD8⁺ T-cell counts after the initial anti-CD8 antibody administration. Group N macaque, R09-009, was administered anti-CD8 antibody at week 108 post-infection and on days 3, 7, and 10 after the first administration. (B) Changes in plasma viral loads. (C) Changes in CD8⁺ T-cell responses specific for SIV Gag, Nef, Vif, Vpx, Vpr, Tat, Rev, Pol, and Env. (D) CD8⁺ T-cell responses specific for SIV Gag_206–216, Gag_241–249, Gag_367–381, Vif_114–124, Nef_9–19, Nef_89–97, and Nef_193–203 epitopes at week 113 post-infection. (E) Dominant non-synonymous mutations in plasma viral cDNA regions encoding Gag, Vif, and Nef epitopes. Viral <i>gag</i>, <i>vif</i>, and <i>nef</i> cDNA fragments were amplified from plasma RNA obtained at weeks 110 and 118 post-infection. Amino acid substitutions around SIV Gag_206–216, Gag_241–249, Gag_367–381, Vif_114–124, Nef_9–19, Nef_89–97, and Nef_193–203 epitopes are shown.</p