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SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication.

ABSTRACT: 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.

SUBMITTER: Neuvonen M

PROVIDER: S-EPMC3219718 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication.

Neuvonen Maarit M Kazlauskas Arunas A Martikainen Miika M Hinkkanen Ari A Ahola Tero T Saksela Kalle K

PLoS pathogens 20111117 11

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 ...[more]

PMID: 22114558

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Project description:Alphaviruses are widely distributed in both hemispheres and circulate between mosquitoes and amplifying vertebrate hosts. Geographically separated alphaviruses have adapted to replication in particular organisms. The accumulating data suggest that this adaptation is determined not only by changes in their glycoproteins but also by the amino acid sequence of the hypervariable domain (HVD) of the alphavirus nsP3 protein. We performed a detailed investigation of chikungunya virus (CHIKV) nsP3 HVD interactions with host factors and their roles in viral replication in vertebrate and mosquito cells. The results demonstrate that CHIKV HVD is intrinsically disordered and binds several distinctive cellular proteins. These host factors include two members of the G3BP family and their mosquito homolog Rin, two members of the NAP1 family, and several SH3 domain-containing proteins. Interaction with G3BP proteins or Rin is an absolute requirement for CHIKV replication, although it is insufficient to solely drive it in either vertebrate or mosquito cells. To achieve a detectable level of virus replication, HVD needs to bind members of at least one more protein family in addition to G3BPs. Interaction with NAP1L1 and NAP1L4 plays a more proviral role in vertebrate cells, while binding of SH3 domain-containing proteins to a proline-rich fragment of HVD is more critical for virus replication in the cells of mosquito origin. Modifications of binding sites in CHIKV HVD allow manipulation of the cell specificity of CHIKV replication. Similar changes may be introduced into HVDs of other alphaviruses to alter their replication in particular cells or tissues.IMPORTANCE Alphaviruses utilize a broad spectrum of cellular factors for efficient formation and function of replication complexes (RCs). Our data demonstrate for the first time that the hypervariable domain (HVD) of chikungunya virus nonstructural protein 3 (nsP3) is intrinsically disordered. It binds at least 3 families of cellular proteins, which play an indispensable role in viral RNA replication. The proteins of each family demonstrate functional redundancy. We provide a detailed map of the binding sites on CHIKV nsP3 HVD and show that mutations in these sites or the replacement of CHIKV HVD by heterologous HVD change cell specificity of viral replication. Such manipulations with alphavirus HVDs open an opportunity for development of new irreversibly attenuated vaccine candidates. To date, the disordered protein fragments have been identified in the nonstructural proteins of many other viruses. They may also interact with a variety of cellular factors that determine critical aspects of virus-host interactions.

Project description:Alphaviruses are enveloped, positive-sense RNA viruses that are important causes of viral encephalomyelitis. Sindbis virus (SINV) infects the neurons of rodents and is a model for studying factors that regulate infection of neuronal cells. The outcome of alphavirus infection of the central nervous system is dependent on neuronal maturation status. Differentiated mature neurons survive and control viral replication better than undifferentiated immature neurons. The cellular factors involved in age-dependent susceptibility include higher levels of antiapoptotic and innate immune factors in mature neurons. Because NF-?B pathway activation is required for the initiation of both apoptosis and the host antiviral response, we analyzed the role of NF-?B during SINV infection of differentiated and undifferentiated rat neuronal cells. SINV infection induced canonical NF-?B activation, as evidenced by the degradation of I?B? and the phosphorylation and nuclear translocation of p65. Inhibition or deletion of the upstream I?B kinase substantially reduced SINV replication in differentiated but not in undifferentiated neuronal cells or mouse embryo fibroblasts. NF-?B inhibition did not affect the establishment of infection, replication complex formation, the synthesis of nonstructural proteins, or viral RNA synthesis in differentiated neurons. However, the translation of structural proteins was impaired, phosphorylation of the ? subunit of eukaryotic translation initiation factor 2 (eIF2?) was decreased, and host protein synthesis was maintained, suggesting that NF-?B activation was involved in the regulation of translation during infection of mature neurons. Inhibition or deletion of double-stranded RNA-activated protein kinase (PKR) also decreased eIF2? phosphorylation, the translation of viral structural proteins, and virus production. Therefore, canonical NF-?B activation synergizes with PKR to promote SINV replication in differentiated neurons by facilitating viral structural protein translation.IMPORTANCE Mosquito-borne alphaviruses are a significant and growing cause of viral encephalomyelitis worldwide. The outcome of alphaviral neuronal infections is host age dependent and greatly affected by neuronal maturation status, with differentiated, mature neurons being more resistant to infection than undifferentiated, immature neurons. The biological factors that change during neuronal maturation and that influence the outcome of viral infection are currently only partially defined. These studies investigated the role of NF-?B in determining the outcome of alphaviral infection in mature and immature neurons. Inhibition of canonical NF-?B activation decreased alphavirus replication in mature neurons by regulating protein synthesis and limiting the production of the viral structural proteins but had little effect on viral replication in immature neurons or fibroblasts. Therefore, NF-?B is a signaling pathway that influences the maturation-dependent outcome of alphaviral infection in neurons and that highlights the importance of cellular context in determining the effects of signal pathway activation.

Dataset Information

SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication.

Publications

SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication.

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