Troyer syndrome is an autosomal recessive hereditary spastic paraplegia caused by mutation in the spartin (SPG20) gene, which encodes a widely expressed protein of unknown function. This mutation results in premature protein truncation and thus might signify a loss-of-function disease mechanism. In this study, we have found that spartin is monoubiquitinated and functions in degradation of the epidermal growth factor receptor (EGFR). Upon EGF stimulation, spartin translocates from the cytoplasm to the plasma membrane and colocalizes with internalized EGF-Alexa. Knockdown of spartin by small interfering RNA decreases the rate of EGFR degradation and also affects EGFR internalization, recycling, or both. Furthermore, overexpression of spartin results in a prominent decrease in EGFR degradation. Taken together, our data suggest that spartin is involved in the intracellular trafficking of EGFR and that impaired endocytosis may underlie the pathogenesis of Troyer syndrome. INTRODUCTIONThe hereditary spastic paraplegias (HSPs) comprise a cluster of inherited neurological disorders characterized by progressive spasticity and muscle weakness in the lower limbs Reid, 2003;Fink, 2006;Soderblom and Blackstone, 2006). Classically, the HSPs have been divided into two forms: "pure" when lower extremity spasticity and paraparesis are the only features and "complicated" when additional symptoms are present (Harding, 1983). More than 30 genetic loci (SPG1-33) and 14 gene products have been identified, yielding new insights into the molecular pathways involved in the pathogenesis of the HSPs. Troyer syndrome (SPG20), an autosomal recessive, complicated HSP that manifests in early childhood, is characterized by dysarthria, mental retardation, shortness of stature, and distal muscle wasting in addition to spasticity and weakness of the lower limbs (Cross and McKusick, 1967;Proukakis et al., 2004). To date, the only known mutation involved in Troyer syndrome is a single base deletion in the spartin gene, resulting in a 29-amino acid substitution at the C-terminus and premature truncation of the 666-amino acid protein by 268 residues (Patel et al., 2002;Proukakis et al., 2004).Although the cellular functions of spartin are not known, it harbors two conserved domains, an MIT (contained within microtubule-interacting and trafficking molecules) domain at the N-terminus and a plant-related region at the C-terminus. Although the only known function of the latter domain is in senescence (Panavas et al., 1999), the MIT domain is found in several other proteins, including Vps4-A and -B, sortin 15, and spastin (Ciccarelli et al., 2003), which are involved in vesicle trafficking and binding of microtubules. Interestingly, spastin, a microtubule-severing ATPase (Errico et al., 2002;Trotta et al., 2004;Evans et al., 2005), is mutated in the most common form of autosomal dominant HSP, SPG4 Reid, 2003;Fink, 2006;Soderblom and Blackstone, 2006). Although a region adjacent to the MIT domain is involved in the interaction of spastin with microtubules ...

BackgroundSince its emergence in 2007 in Micronesia and Polynesia, the arthropod-borne flavivirus Zika virus (ZIKV) has spread in the Americas and the Caribbean, following first detection in Brazil in May 2015. The risk of ZIKV emergence in Europe increases as imported cases are repeatedly reported. Together with chikungunya virus (CHIKV) and dengue virus (DENV), ZIKV is transmitted by Aedes mosquitoes. Any countries where these mosquitoes are present could be potential sites for future ZIKV outbreak. We assessed the vector competence of European Aedes mosquitoes (Aedes aegypti and Aedes albopictus) for the currently circulating Asian genotype of ZIKV.Methodology/Principal FindingsTwo populations of Ae. aegypti from the island of Madeira (Funchal and Paul do Mar) and two populations of Ae. albopictus from France (Nice and Bar-sur-Loup) were challenged with an Asian genotype of ZIKV isolated from a patient in April 2014 in New Caledonia. Fully engorged mosquitoes were then maintained in insectary conditions (28°±1°C, 16h:8h light:dark cycle and 80% humidity). 16–24 mosquitoes from each population were examined at 3, 6, 9 and 14 days post-infection to estimate the infection rate, disseminated infection rate and transmission efficiency. Based on these experimental infections, we demonstrated that Ae. albopictus from France were not very susceptible to ZIKV.Conclusions/SignificanceIn combination with the restricted distribution of European Ae. albopictus, our results on vector competence corroborate the low risk for ZIKV to expand into most parts of Europe with the possible exception of the warmest regions bordering the Mediterranean coastline.

The viral determinants of Alphavirus-induced rheumatic disease have not been elucidated. We identified an RRV strain (DC5692) which, in contrast to the T48 strain, does not induce musculoskeletal inflammation in a mouse model of RRV disease. Substitution of the RRV T48 strain nonstructural protein 1 (nsP1) coding sequence with that from strain DC5692 generated a virus that was attenuated in vivo despite similar viral loads in tissues. In contrast, substitution of the T48 PE2 coding region with the PE2 coding region from DC5692 resulted in attenuation in vivo and reduced viral loads in tissues. In gain of virulence experiments, substitution of the DC5692 strain nsP1 and PE2 coding regions with those from the T48 strain was sufficient to restore full virulence to the DC5692 strain. These findings indicate that determinants in both nsP1 and PE2 have critical and distinct roles in the pathogenesis of RRV-induced musculoskeletal inflammatory disease in mice.

Ross River virus (RRV) is one of a group of mosquito-transmitted alphaviruses that cause debilitating, and often chronic, musculoskeletal disease in humans. Previously, we reported that replacement of the nonstructural protein 1 (nsP1) gene of the mouse-virulent RRV strain T48 with that from the mouse-avirulent strain DC5692 generated a virus that was attenuated in a mouse model of disease. Here we find that the six nsP1 nonsynonymous nucleotide differences between strains T48 and DC5692 are determinants of RRV virulence, and we identify two nonsynonymous nucleotide changes as sufficient for the attenuated phenotype. RRV T48 carrying the six nonsynonymous DC5692 nucleotide differences (RRV-T48-nsP1 6M ) was attenuated in both wild-type and Rag1 ؊/؊ mice. Despite the attenuated phenotype, RRV T48 and RRV-T48-nsP1 6M loads in tissues of wild-type and Rag1 ؊/؊ mice were indistinguishable from 1 to 3 days postinoculation. RRV-T48-nsP1 6M loads in skeletal muscle tissue, but not in other tissues, decreased dramatically by 5 days postinoculation in both wild-type and Rag1 ؊/؊ mice, suggesting that the RRV-T48-nsP1 6M mutant is more sensitive to innate antiviral effectors than RRV T48 in a tissue-specific manner. In vitro, we found that the attenuating mutations in nsP1 conferred enhanced sensitivity to type I interferon. In agreement with these findings, RRV T48 and RRV-T48-nsP1 6M loads were similar in mice deficient in the type I interferon receptor. Our findings suggest that the type I IFN response controls RRV infection in a tissue-specific manner and that specific amino acid changes in nsP1 are determinants of RRV virulence by regulating the sensitivity of RRV to interferon. IMPORTANCEArthritogenic alphaviruses, including Ross River virus (RRV), infect humans and cause debilitating pain and inflammation of the musculoskeletal system. In this study, we identified coding changes in the RRV nsP1 gene that control the virulence of RRV and its sensitivity to the antiviral type I interferon response, a major component of antiviral defense in mammals. Furthermore, our studies revealed that the effects of these attenuating mutations are tissue specific. These findings suggest that these mutations in nsP1 influence the sensitivity of RRV to type I interferon only in specific host tissues. The new knowledge gained from these studies contributes to our understanding of host responses that control alphavirus infection and viral determinants that counteract these responses.