SH3 Domain-Mediated Recruitment of Host Cell Amphiphysins by Alphavirus nsP3 Promotes Viral RNA Replication

et al. (2011) SH3 Domain-Mediated Recruitment of Host Cell Amphiphysins by Alphavirus nsP3 Promotes Viral RNA Replication. PLoS Pathog 7(11): e1002383. doi:10.1371/journal.ppat.1002383 SH3 Domain-Mediated Recruitment of Host Cell Amphiphysins by Alphavirus nsP3 Promotes Viral RNA Replication Maarit Neuvonen 0 Arunas Kazlauskas 0 Miika Martikainen 0 Ari Hinkkanen 0 Tero Ahola 0 Kalle 0 Saksela 0 Felix A. Rey, Institut Pasteur, France 0 1 Institute of Biotechnology, University of Helsinki , Helsinki , Finland , 2 Department of Virology, Haartman Institute, University of Helsinki and Helsinki University Central Hospital , Helsinki , Finland , 3 A. I. Virtanen Institute, Department of Biotechnology and Molecular Medicine, University of Eastern Finland and the Cancer Center of Eastern Finland , Kuopio , Finland Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3) domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV), Sindbis (SINV), and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication. - Funding: This study was supported by grants from the Academy of Finland (127197 to KS, 127214 to TA, and 137958 to AH), Helsinki University Central Hospital Research Council (TYH2008307 to KS), the Sigrid Juselius Foundation to KS and TA, and the strategic funding of the University of Eastern Finland to AH. MN was supported in part by a fellowship from the Viikki Doctoral Programme in Molecular Biosciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. The genus Alphavirus (family Togaviridae) includes some 30 known members. The alphaviruses are enveloped positive-strand RNA viruses with a 59 capped and 39 polyadenylated genome of approximately 11.5 kb. Most alphaviruses are mosquito-borne viruses, and some are capable of causing serious disease in humans and domestic animals [1,2]. On the American continents, Venezuelan, Western, and Eastern equine encephalitis viruses occasionally cause epidemics in horses, which can also spill over to infect humans with potentially lethal consequences. In contrast, Old World alphaviruses, including Ross River virus and Sindbis virus (SINV), are associated with fever, rash and painful, debilitating arthritis, which can persist for months or even years. Most recently, starting in 2005, Chikungunya virus (CHKV) reemerged to cause a large epidemic around the Indian Ocean, infecting approximately 10 million people [3]. Alphavirus RNA replication takes place in small membrane invaginations that protrude from the inner surface of the host cell plasma membrane and from the outer surface of endosomes and lysosomes [4]. In infected cells the endo-lysosomes are ultrastructurally altered by the viral replication complexes, and are termed cytopathic vacuoles type I (CPVs) [5]. The replication complexes contain as essential components the virus-encoded nonstructural proteins nsP1-nsP4, which arise through cleavage from a polyprotein precursor P1234. NsP1, nsP2 and nsP4 possess essential enzymatic activities of RNA capping, helicase/protease, and polymerase, respectively (for a review see [6]). The functions of nsP3 have remained more obscure, although mutations in it affect various steps of RNA synthesis [7]. The Nterminus of nsP3 contains a structurally conserved protein domain termed the macro domain, which is capable of binding ADP-ribose derivatives and RNA, and also hydrolyzing ADP-ribose-199phosphate [8,9]. Although the roles of these activities in RNA replication remain to be clarified, mutations in the macro domain affect RNA synthesis [10]. The C-terminus of alphavirus nsP3 contains a tail region, which varies in length between ,150250 amino acid residues in different alphavirus and is devoid of predicted secondary structure. Interestingly, the tail is hypervariable, showing no overall sequence conservation even between related alphaviruses. Nevertheless, the tail region has been implicated in the virulence of alphaviruses [11]. Some regions of the tail are also heavily phosphorylated on serine and threonine residues, and in Semliki Forest virus (SFV), deletion of the The genus Alphavirus contains 29 known species that are transmitted by arthropods and include many important pathogens, such as Chikungunya virus (CHKV), which during the past decade has re-emerged to cause massive epidemics of febrile arthralgia around the Indian Ocean. The role of the alphaviral non-structural protein 3 (nsP3) has been linked to RNA replication and disease pathogenesis, but its molecular functions have remained elusive. Here we show that the nsP3s of CHKV as well as Sindbis and Semliki Forest viruses use a conserved proline-rich motif to interact with the Src-homology-3 (SH3) domain of host cell amphiphysins Amph1 and BIN1/Amph2, two adaptor proteins prominently involved in cellular membrane dynamics. We observed a striking re-localization of amphiphysin to alphaviral replication complexes in infected cells, and found that disruption of the amphiphysin SH3 binding motif in nsP3 strongly suppressed virus replication in vitro and attenuated Semliki Forest virus in infected mice. Thus, we conclude that amphiphysins are novel and important host cell factors involved in supporting alphavirus replication. phosphorylated region gave rise to a virus that replicated well in cell culture but was apathogenic in mice [12]. Multiple host proteins associated with nsP3 have been identified via immunoprecipitation and mass spectrometry [13,14], but the significance of these interactions as well as the relevant protein binding sites involved have remained uncharacterized. In addition to being present in the replication complexes in the CPVs and at the plasma membrane, a large fraction (...truncated)