A structural constraint for functional interaction between N-terminal and C-terminal domains in simian immunodeficiency virus capsid proteins

<p>Abstract</p> <p>Background</p> <p>The Gag capsid (CA) is one of the most conserved proteins in highly-diversified human and simian immunodeficiency viruses (HIV and SIV). Understanding the limitations imposed on amino acid sequences in CA could provide valuable information for vaccine immunogen design or anti-HIV drug development. Here, by comparing two pathogenic SIV strains, SIVmac239 and SIVsmE543-3, we found critical amino acid residues for functional interaction between the N-terminal and the C-terminal domains in CA.</p> <p>Results</p> <p>We first examined the impact of Gag residue 205, aspartate (Gag205D) in SIVmac239 and glutamate (Gag205E) in SIVsmE543-3, on viral replication; due to this difference, Gag_206-216(IINEEAADWDL) epitope-specific cytotoxic T lymphocytes (CTLs) were previously shown to respond to SIVmac239 but not SIVsmE543-3 infection. A mutant SIVmac239, SIVmac239Gag205E, whose Gag205D is replaced with Gag205E showed lower replicative ability. Interestingly, however, SIVmac239Gag205E passaged in macaque T cell culture often resulted in selection of an additional mutation at Gag residue 340, a change from SIVmac239 valine (Gag340V) to SIVsmE543-3 methionine (Gag340M), with recovery of viral fitness. Structural modeling analysis suggested possible intermolecular interaction between the Gag205 residue in the N-terminal domain and Gag340 in the C-terminal in CA hexamers. The Gag205D-to-Gag205E substitution in SIVmac239 resulted in loss of in vitro core stability, which was recovered by additional Gag340V-to-Gag340M substitution. Finally, selection of Gag205E plus Gag340M mutations, but not Gag205E alone was observed in a chronically SIVmac239-infected rhesus macaque eliciting Gag_206-216-specific CTL responses.</p> <p>Conclusions</p> <p>These results present in vitro and in vivo evidence implicating the interaction between Gag residues 205 in CA NTD and 340 in CA CTD in SIV replication. Thus, this study indicates a structural constraint for functional interaction between SIV CA NTD and CTD, providing insight into immunogen design to limit viral escape options.</p

Truncated KCNQ1 mutant, A178fs/105, forms hetero-multimer channel with wild-type causing a dominant-negative suppression due to trafficking defect

AbstractWe identified a novel mutation Ala178fs/105 missing S3–S6 and C-terminus portions of KCNQ1 channel. Ala178fs/105-KCNQ1 expressed in COS-7 cells demonstrated no current expression. Co-expression with wild-type (WT) revealed a dominant-negative effect, which suggests the formation of hetero-multimer by mutant and WT. Confocal laser microscopy displayed intracellular retention of Ala178fs/105-KCNQ1 protein. Co-expression of the mutant and WT also increased intracellular retention of channel protein compared to WT alone. Our findings suggest a novel mechanism for LQT1 that the truncated S1–S2 KCNQ1 mutant forms hetero-multimer and cause a dominant-negative effect due to trafficking defect

Tcap gene mutations in hypertrophic cardiomyopathy and dilated cardiomyopathy

ObjectivesWe sought to explore the relationship between a Tcap gene (TCAP)abnormality and cardiomyopathy.BackgroundHypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) cause severe heart failure and sudden death. Recent genetic investigations have revealed that mutations of genes encoding Z-disc components, including titin and muscle LIM protein (MLP), are the primary cause of both HCM and DCM. The Z-disc plays a role in establishing the mechanical coupling of sarcomeric contraction and stretching, with the titin/Tcap/MLP complex serving as a mechanical stretch sensor. Tcap interacts with the calsarcin, which tethers the calcineurin to the Z-disc.MethodsThe TCAPwas analyzed in 346 patients with HCM (236 familial and 110 sporadic cases) and 136 patients with DCM (34 familial and 102 sporadic cases). Two different in vitro qualitative assays—yeast two-hybrid and glutathion S-transferase pull-down competition—were performed in order to investigate functional changes in Tcap's interaction with MLP, titin, and calsarcin-1 caused by the identified mutations and a reported DCM-associated mutation, R87Q.ResultsTwo TCAPmutations, T137I and R153H, were found in patients with HCM, and another TCAPmutation, E132Q, was identified in a patient with DCM. It was demonstrated by the qualitative assays that the HCM-associated mutations augment the ability of Tcap to interact with titin and calsarcin-1, whereas the DCM-associated mutations impair the interaction of Tcap with MLP, titin, and calsarcin-1.ConclusionsThese observations suggest that the difference in clinical phenotype (HCM or DCM) may be correlated with the property of altered binding among the Z-disc components

Host cell species-specific effect of cyclosporine A on simian immunodeficiency virus replication

<p>Abstract</p> <p>Background</p> <p>An understanding of host cell factors that affect viral replication contributes to elucidation of the mechanism for determination of viral tropism. Cyclophilin A (CypA), a peptidyl-prolyl <it>cis-trans </it>isomerase (PPIase), is a host factor essential for efficient replication of human immunodeficiency virus type 1 (HIV-1) in human cells. However, the role of cyclophilins in simian immunodeficiency virus (SIV) replication has not been determined. In the present study, we examined the effect of cyclosporine A (CsA), a PPIase inhibitor, on SIV replication.</p> <p>Results</p> <p>SIV replication in human CEM-SS T cells was not inhibited but rather enhanced by treatment with CsA, which inhibited HIV-1 replication. CsA treatment of target human cells enhanced an early step of SIV replication. CypA overexpression enhanced the early phase of HIV-1 but not SIV replication, while CypA knock-down resulted in suppression of HIV-1 but not SIV replication in CEM-SS cells, partially explaining different sensitivities of HIV-1 and SIV replication to CsA treatment. In contrast, CsA treatment inhibited SIV replication in macaque T cells; CsA treatment of either virus producer or target cells resulted in suppression of SIV replication. SIV infection was enhanced by CypA overexpression in macaque target cells.</p> <p>Conclusions</p> <p>CsA treatment enhanced SIV replication in human T cells but abrogated SIV replication in macaque T cells, implying a host cell species-specific effect of CsA on SIV replication. Further analyses indicated a positive effect of CypA on SIV infection into macaque but not into human T cells. These results suggest possible contribution of CypA to the determination of SIV tropism.</p

Recent advances in micro-sized oxygen carriers inspired by red blood cells

ABSTRACTSupplementing sufficient oxygen to cells is always challenging in biomedical engineering fields such as tissue engineering. Originating from the concept of a ‘blood substitute’, nano-sized artificial oxygen carriers (AOCs) have been studied for a long time for the optimization of the oxygen supplementation and improvement of hypoxia environments in vitro and in vivo. When circulating in our bodies, micro-sized human red blood cells (hRBCs) feature a high oxygen capacity, a unique biconcave shape, biomechanical and rheological properties, and low frictional surfaces, making them efficient natural oxygen carriers. Inspired by hRBCs, recent studies have focused on evolving different AOCs into microparticles more feasibly able to achieve desired architectures and morphologies and to obtain the corresponding advantages. Recent micro-sized AOCs have been developed into additional categories based on their principal oxygen-carrying or oxygen-releasing materials. Various biomaterials such as lipids, proteins, and polymers have also been used to prepare oxygen carriers owing to their rapid oxygen transfer, high oxygen capacity, excellent colloidal stability, biocompatibility, suitable biodegradability, and long storage. In this review, we concentrated on the fabrication techniques, applied biomaterials, and design considerations of micro-sized AOCs to illustrate the advances in their performances. We also compared certain recent micro-sized AOCs with hRBCs where applicable and appropriate. Furthermore, we discussed existing and potential applications of different types of micro-sized AOCs

Localization of photoperiod responsive circadian oscillators in the mouse suprachiasmatic nucleus

The circadian pacemaker in the suprachiasmatic nucleus (SCN) yields photoperiodic response to transfer seasonal information to physiology and behavior. To identify the precise location involved in photoperiodic response in the SCN, we analyzed circadian Period1 and PERIOD2 rhythms in horizontally sectioned SCN of mice exposed to a long or short day. Statistical analyses of bioluminescence images with respective luciferase reporters on pixel level enabled us to identify the distinct localization of three oscillating regions; a large open-ring-shape region, the region at the posterior end and a sharply demarcated oval region at the center of the SCN. The first two regions are the respective sites for the so-called evening and morning oscillators, and the third region is possibly a site for mediating photic signals to the former oscillators. In these regions, there are two classes of oscillating cells in which Per1 and Per2 could play differential roles in photoperiodic responses

A Case of Primary Ciliary Dyskinesia Caused by a Mutation in OFD1, Which Was Diagnosed Owing to Clostridium difficile Infection

We report a Japanese 5-year-old boy with primary ciliary dyskinesia (PCD) which was diagnosed owing to Clostridium difficile (CD) infection caused by prolonged antibiotic exposure. He had intractable otitis media with effusion (OME) and had abdominal pain and diarrhea for 4 months after starting antibiotics administration. His stool contained CD toxin. After vancomycin treatment, his symptoms improved and his stools did not contain CD toxin. His past medical history included frequent pneumonia. We, therefore, performed electron microscopy of the biopsy specimen from his nasal mucosa and genetic testing, and he was diagnosed with PCD. PCD is a rare inherited genetic disease causing ciliary dysfunction, which is very difficult to diagnose because some children without PCD also develop the same symptoms. Therefore, children who have intractable OME, rhinosinusitis, frequent pneumonia, or bronchitis and are taking antibiotics for long periods of time should be checked for underlying diseases, such as PCD

Solution Structures of Cytosolic RNA Sensor MDA5 and LGP2 C-terminal Domains : Identification of the RNA Recognition Loop in RIG-I-LIKE Receptors

The RIG-I like receptor (RLR) comprises three homologues: RIG-I (retinoic acid-inducible gene I), MDA5(melanoma differentiation-associated gene 5), and LGP2 (laboratory of genetics and physiology 2). Each RLR senses different viral infections by recognizing replicating viral RNA in the cytoplasm. The RLR contains a conserved C-terminal domain (CTD), which is responsible for the binding specificity to the viral RNAs, including double-stranded RNA (dsRNA) and 5'-triphosphated single-stranded RNA (5'ppp-ssRNA). Here, the solution structures of the MDA5 and LGP2 CTD domains were solved by NMR and compared with those of RIG-I CTD. The CTD domains each have a similar fold and a similar basic surface but there is the distinct structural feature of a RNA binding loop; The LGP2 and RIG-I CTD domains have a large basic surface, one bank of which is formed by the RNA binding loop. MDA5 also has a large basic surface that is extensively flat due to open conformation of the RNA binding loop. The NMR chemical shift perturbation study showed that dsRNA and 5'ppp-ssRNA are bound to the basic surface of LGP2 CTD, whereas dsRNA is bound to the basic surface of MDA5 CTD but much more weakly, indicating that the conformation of the RNA binding loop is responsible for the sensitivity to dsRNA and 5'ppp-ssRNA. Mutation study of the basic surface and the RNA binding loop supports the conclusion from the structure studies. Thus, the CTD is responsible for the binding affinity to the viral RNAs