SMA patient-derived mutations show a range of life expectancies in <i>Drosophila</i>.

<p>(A) Viability analyses of SMA point mutations in <i>Drosophila</i>. <i>Smn^X7/TM6.tb GFP</i> flies were crossed with each mutant line (<i>Smn^X7,Smn^Tg/TM6.tb GFP</i>) and hemizygous first instar mutant larvae (<i>Smn^X7/X7, Smn^Tg/−</i> where <i>Tg</i> stands for <i>transgene</i>) were collected and followed through development. Oregon-R (OR) animals served as controls. The data for each genotype are expressed as a fraction of pupae or adults over the total number of starting larvae (n), listed below each genotype. Expression of the WT transgene (<i>Smn^X7/X7,Smn^WT/−</i>) shows robust rescue of the null phenotype (∼67% adults). The mutants show a range in severity, from very severe (larval lethal), to intermediate (pupal lethal), to mild (adults). (B) Expression levels of <i>Smn</i> transgenes were examined by western blotting. Larval lysates from homozygous mutant lines (<i>Smn^X7/X7,Smn^Tg/Tg</i>), were probed with anti-dSMN or anti-FLAG antibodies, as indicated. The slower migrating bands represent the FLAG-tagged transgenic proteins and the faster migrating band corresponds to endogenous dSMN, present only in the OR controls. The mutant transgenes show similar levels of dSMN protein compared to <i>Smn^WT</i> animals, with the exception of <i>Smn^M194R, Smn^Y203C</i> and <i>Smn^G206S</i>, which show a significant reduction. Tubulin was used as a loading control.</p

SMA-Causing Missense Mutations in <i>Survival motor neuron (Smn)</i> Display a Wide Range of Phenotypes When Modeled in <i>Drosophila</i>

<div><p>Mutations in the human <i>survival motor neuron 1</i> (<i>SMN</i>) gene are the primary cause of spinal muscular atrophy (SMA), a devastating neuromuscular disorder. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. Additional tissue-specific and global functions have been ascribed to SMN; however, their relevance to SMA pathology is poorly understood and controversial. Using <i>Drosophila</i> as a model system, we created an allelic series of twelve <i>Smn</i> missense mutations, originally identified in human SMA patients. We show that animals expressing these SMA-causing mutations display a broad range of phenotypic severities, similar to the human disease. Furthermore, specific interactions with other proteins known to be important for SMN's role in RNP assembly are conserved. Intragenic complementation analyses revealed that the three most severe mutations, all of which map to the YG box self-oligomerization domain of SMN, display a stronger phenotype than the null allele and behave in a dominant fashion. In support of this finding, the severe YG box mutants are defective in self-interaction assays, yet maintain their ability to heterodimerize with wild-type SMN. When expressed at high levels, wild-type SMN is able to suppress the activity of the mutant protein. These results suggest that certain SMN mutants can sequester the wild-type protein into inactive complexes. Molecular modeling of the SMN YG box dimer provides a structural basis for this dominant phenotype. These data demonstrate that important structural and functional features of the SMN YG box are conserved between vertebrates and invertebrates, emphasizing the importance of self-interaction to the proper functioning of SMN.</p></div

Incomplete dominance of <i>Smn</i> mutants.

<p>(A) Each of the <i>Smn</i> point mutant lines (<i>Smn^X7,Smn^Tg/TM6-tb.GFP</i>) was crossed to the wild-type (WT) rescue line (<i>Smn^WT</i>,<i>Smn^WT/TM6-tb.GFP</i>). For most mutations, this resulted in an intermediate level of rescue, between that observed when expressing the wild-type transgene alone versus that of the mutant transgenes alone. <i>Smn^X7/X7</i>,<i>Smn^Tg/WT</i> flies (WT) are shown in yellow and <i>Smn^X7/X7</i>,<i>Smn^Tg/−</i> flies (X7) are shown in purple. Genotypes of the various transgenes are listed along the X-axis. Larval progeny were followed through development and the number of eclosing adult flies was measured and expressed as a fraction of the total number of starting animals (n) for each genotype. (B) The <i>Smn^X7,Smn^Y203C</i> mutants were crossed with each of the other transgenic lines, both mutant (<i>Smn^X7,Smn^Tg</i>) and wild-type (<i>Smn^X7,Smn^WT</i>). Larval progeny were followed through development and the number of pupae and adults was measured and expressed as a fraction of the total number of starting animals (n) for each genotype. (C) The <i>Smn^X7,Smn^G210V</i> mutants were crossed to the other alleles, as described in panel B.</p

<i>SMN^Y203C</i> is predicted to disrupt an intermolecular Y-G contact, weakening or preventing oligomerization.

<p>A combination of experimental analysis and structural modeling of <i>Drosophila</i> SMN dimers reveals that wild-type homodimers (left column) can make two Y-G contacts and are able to form higher-order structures (active SMN oligomers). In contrast, Y203C homodimers (right) are unable to make these contacts and do not dimerize (inactive SMN monomers). Interestingly, Y203C:WT heterodimers (middle) make only one Y-G contact but the loss of one interaction does not prevent dimerization. These findings suggest that the dominant negative activity of Y203C is due to an inability of the heterodimers to form active, higher-order oligomers.</p

Phenotypic characterization of three severe YG box mutants.

<p>(A) Viability of larvae hemizygous for the <i>Smn^M194R, Smn^G206S and Smn^Y203C</i> mutations was assayed. The graph tracks survival of approximately100 larvae (n) for each genotype (<i>Smn^X7/X7,Smn^Tg/−</i>), along with homozygous null mutant (<i>Smn^X7/Smn^X7</i>) and Oregon-R (OR) controls, over time. All three missense mutants die earlier than the null animals, suggesting a dominant negative effect. Note that by day 5 all of the wild-type control larvae have pupated. (B) Graph of average distance traveled over 20 s after stimulation with a needle (n>10 larvae were scored for each genotype). Larval movement was impaired for <i>M194R, Y203C and G206S</i> larvae, relative to <i>WT</i> transgene controls (P<1.3×10⁻⁵). (C) Graph of overall larval size, as measured by area (in mm²). For each genotype, n>24 larvae were scored (P<1.8×10⁻⁷). For illustration, an image of two representative larvae (<i>Smn^WT</i> and <i>Smn^G206S</i>) is shown.</p

An allelic series of SMN missense mutations in <i>Drosophila</i>.

<p>(A) Alignment of SMN orthologues from <i>Homo sapiens</i> (Hs), <i>Danio Rerio</i> (Dr), <i>Apis Mellifera</i> (Am), and <i>Drosophila melanogaster</i> (Dm). Conserved regions, including the Gemin2 binding domain, the Tudor domain and the YG box of SMN, are shaded in gray. Residues known to be mutated in SMA patients are highlighted in aqua. SMA-causing missense residues (SMA) are listed below the highlighted residues. (B) Summary of the human SMA patient mutations that were modeled in <i>Drosophila</i>. These mutations affect all major domains of SMN, and encompass all three classes of SMA severity, with Type I being the most severe and Type III being the mildest form. When multiple SMA types are listed for a given mutation, this indicates that differing phenotypic severities were observed in patients bearing the same mutation.</p

Interaction of SMN mutants with proteins involved in RNP assembly.

<p>(A) <i>Smn^M194R</i> and <i>Smn^G206S</i> are defective in a self-oligomerization assay. Note that oligomerization of <i>Smn^Y203C</i> and <i>Smn^T205I</i> was tested previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004489#pgen.1004489-Praveen2" target="_blank">[28]</a>. Lysates were prepared from cells co-expressing FLAG- and Myc-tagged versions of dSMN mutants or WT controls. Co-immunoprecipitation was performed with anti-FLAG antibody followed by western analysis with anti-Myc antibody to visualize the amount of Myc-tagged dSMN that co-precipitated with the FLAG-tagged protein. (B) dSMN(D20V) protein shows defective binding to dGem2. Immunoprecipitation was performed with anti-FLAG antibody followed by western analysis with anti-Myc antibody to visualize the amount of Myc-tagged dGem2. (C) dSMN(Y203C) and dSMN(G206S) are defective in their ability to bind dGem3. Immunoprecipitation of Myc-dGem3 was followed by probing with anti-FLAG antibody to visualize Flag-dSMN.</p

Structural Basis for Inhibitor-Induced Aggregation of HIV Integrase

<div><p>The allosteric inhibitors of integrase (termed ALLINIs) interfere with HIV replication by binding to the viral-encoded integrase (IN) protein. Surprisingly, ALLINIs interfere not with DNA integration but with viral particle assembly late during HIV replication. To investigate the ALLINI inhibitory mechanism, we crystallized full-length HIV-1 IN bound to the ALLINI GSK1264 and determined the structure of the complex at 4.4 Å resolution. The structure shows GSK1264 buried between the IN C-terminal domain (CTD) and the catalytic core domain. In the crystal lattice, the interacting domains are contributed by two different dimers so that IN forms an open polymer mediated by inhibitor-bridged contacts; the N-terminal domains do not participate and are structurally disordered. Engineered amino acid substitutions at the inhibitor interface blocked ALLINI-induced multimerization. HIV escape mutants with reduced sensitivity to ALLINIs commonly altered amino acids at or near the inhibitor-bound interface, and these substitutions also diminished IN multimerization. We propose that ALLINIs inhibit particle assembly by stimulating inappropriate polymerization of IN via interactions between the catalytic core domain and the CTD and that understanding the interface involved offers new routes to inhibitor optimization.</p></div

Allosteric inhibitors of HIV IN.

<p>(A) Domain organization of IN. (B) Chemical structures of the ALLINIs used in this study. (C) Disruption of assembly by the ALLINIs GSK1264 and GSK002. Viral particles produced in the presence of 1,000 nM GSK1264 or GSK002 were visualized by transmission electron microscopy, and morphology was scored (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002584#pbio.1002584.s001" target="_blank">S1 Data</a>). The <i>p</i>-value is the probability of obtaining the observed (or greater) differences in numbers of nonmature particles (immature, deformed, or ambiguous) between treated and nontreated samples, given the null hypothesis of no inhibitor-induced changes.</p

HIV IN mutations conferring reduced sensitivity to GSK1264 and GSK002 after serial passage.

<p>(A) Amino acid substitutions resulting from resistance mutations identified by serial passage in the presence of GSK1264 (blue spheres). (B) Substitutions identified in the presence of GSK 002 (orange spheres). Bound GSK1264 is shown in red. (C) The A205T resistance substitutions, which arose in both experiments. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002584#pbio.1002584.s005" target="_blank">S4 Fig</a> provide structural insights into the mechanism of resistance conferred by polymorphisms at residues 124 and 125.</p