Distinct nucleic acid interaction properties of HIV-1 nucleocapsid protein precursor NCp15 explain reduced viral infectivity.

During human immunodeficiency virus type 1 (HIV-1) maturation, three different forms of nucleocapsid (NC) protein-NCp15 (p9 + p6), NCp9 (p7 + SP2) and NCp7-appear successively. A mutant virus expressing NCp15 shows greatly reduced infectivity. Mature NCp7 is a chaperone protein that facilitates remodeling of nucleic acids (NAs) during reverse transcription. To understand the strict requirement for NCp15 processing, we compared the chaperone function of the three forms of NC. NCp15 anneals tRNA to the primer-binding site at a similar rate as NCp7, whereas NCp9 is the most efficient annealing protein. Assays to measure NA destabilization show a similar trend. Dynamic light scattering studies reveal that NCp15 forms much smaller aggregates relative to those formed by NCp7 and NCp9. Nuclear magnetic resonance studies suggest that the acidic p6 domain of HIV-1 NCp15 folds back and interacts with the basic zinc fingers. Neutralizing the acidic residues in p6 improves the annealing and aggregation activity of NCp15 to the level of NCp9 and increases the protein-NA aggregate size. Slower NCp15 dissociation kinetics is observed by single-molecule DNA stretching, consistent with the formation of electrostatic inter-protein contacts, which likely contribute to the distinct aggregate morphology, irregular HIV-1 core formation and non-infectious virus.

Modeling the human MTM1 p.R69C mutation in murine Mtm1 results in exon 4 skipping and a less severe myotubular myopathy phenotype.

X-linked myotubular myopathy (MTM) is a severe neuromuscular disease of infancy caused by mutations of MTM1, which encodes the phosphoinositide lipid phosphatase, myotubularin. The Mtm1 knockout (KO) mouse has a severe phenotype and its short lifespan (8 weeks) makes it a challenge to use as a model in the testing of certain preclinical therapeutics. Many MTM patients succumb early in life, but some have a more favorable prognosis. We used human genotype-phenotype correlation data to develop a myotubularin-deficient mouse model with a less severe phenotype than is seen in Mtm1 KO mice. We modeled the human c.205C>T point mutation in Mtm1 exon 4, which is predicted to introduce the p.R69C missense change in myotubularin. Hemizygous male Mtm1 p.R69C mice develop early muscle atrophy prior to the onset of weakness at 2 months. The median survival period is 66 weeks. Histopathology shows small myofibers with centrally placed nuclei. Myotubularin protein is undetectably low because the introduced c.205C>T base change induced exon 4 skipping in most mRNAs, leading to premature termination of myotubularin translation. Some full-length Mtm1 mRNA bearing the mutation is present, which provides enough myotubularin activity to account for the relatively mild phenotype, as Mtm1 KO and Mtm1 p.R69C mice have similar muscle phosphatidylinositol 3-phosphate levels. These data explain the basis for phenotypic variability among human patients with MTM1 p.R69C mutations and establish the Mtm1 p.R69C mouse as a valuable model for the disease, as its less severe phenotype will expand the scope of testable preclinical therapies.

A rare case of B-lymphoid blast phase of chronic myeloid leukemia: Diagnostic challenges.

Chronic myeloid leukemia(CML) is characterized by Philadelphia(Ph) chromosome. About 5% of cases are diagnosed in blast phase. We report a case of a 53-year-old female with no significant medical history, in B-lymphoblast crisis. Flow cytometry demonstrated B-lymphoblasts with no myeloid aberrancies, together with immature neutrophils in blood, and B-lymphoblasts in bone marrow. Cytogenetic studies identified Ph+ with complex abnormalities. Molecular analysis showed positive both for p210 and p190 transcripts in blood. ABL1 mutation analysis by Next Generation Sequencing(NGS) detected Thr315Ile mutation, which confers resistance to many tyrosine kinase inhibitors(TKIs). Eight months later she received allogeneic transplant and is doing well.

Pediatric Cheerleading Injuries Treated in Emergency Departments in the United States.

This study investigates the epidemiology of cheerleading injuries to children in the United States. Data were analyzed from the National Electronic Injury Surveillance System for children 5 through 18 years of age treated in US emergency departments for cheerleading injuries from 1990 through 2012. An estimated 497 095 children ages 5 to 18 years were treated in US emergency departments for a cheerleading injury during the 23-year study period, averaging 21 613 injured children per year. From 1990 to 2012, the annual cheerleading injury rate increased significantly by 189.1%; and from 2001 to 2012, the annual rate of cheerleading-related concussion/closed head injury increased significantly by 290.9%. Falls were the most common mechanism of injury (29.4%) and were more likely to lead to hospitalization (relative risk = 2.47; 95% confidence interval = 1.67-3.68) compared with other injury mechanisms. The rising number and rate of pediatric cheerleading injuries underscore the need for increased efforts to prevent these injuries.

Single aromatic residue location alters nucleic acid binding and chaperone function of FIV nucleocapsid protein.

Feline immunodeficiency virus (FIV) is a retrovirus that infects domestic cats, and is an excellent animal model for human immunodeficiency virus type 1 (HIV-1) pathogenesis. The nucleocapsid (NC) protein is critical for replication in both retroviruses. FIV NC has several structural features that differ from HIV-1 NC. While both NC proteins have a single conserved aromatic residue in each of the two zinc fingers, the aromatic residue on the second finger of FIV NC is located on the opposite C-terminal side relative to its location in HIV-1 NC. In addition, whereas HIV-1 NC has a highly charged cationic N-terminal tail and a relatively short C-terminal extension, the opposite is true for FIV NC. To probe the impact of these differences on the nucleic acid (NA) binding and chaperone properties of FIV NC, we carried out ensemble and single-molecule assays with wild-type (WT) and mutant proteins. The ensemble studies show that FIV NC binding to DNA is strongly electrostatic, with a higher effective charge than that observed for HIV-1 NC. The C-terminal basic domain contributes significantly to the NA binding capability of FIV NC. In addition, the non-electrostatic component of DNA binding is much weaker for FIV NC than for HIV-1 NC. Mutation of both aromatic residues in the zinc fingers to Ala (F12A/W44A) further increases the effective charge of FIV NC and reduces its non-electrostatic binding affinity. Interestingly, switching the location of the C-terminal aromatic residue to mimic the HIV-1 NC sequence (N31W/W44A) reduces the effective charge of FIV NC and increases its non-electrostatic binding affinity to values similar to HIV-1 NC. Consistent with the results of these ensemble studies, single-molecule DNA stretching studies show that while WT FIV NC has reduced stacking capability relative to HIV-1 NC, the aromatic switch mutant recovers the ability to intercalate between the DNA bases. Our results demonstrate that altering the position of a single aromatic residue switches the binding mode of FIV NC from primarily electrostatic binding to more non-electrostatic binding, conferring upon it NA interaction properties comparable to that of HIV-1 NC.