SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication. that of another proviral host factor, CD2AP. The structural data also demonstrated that FHL1-HVD interaction is mostly determined by the LIM1 domain of FHL1. However, it does not mirror binding of the entire protein, suggesting that other LIM domains are involved. In agreement with previously published data, our biological experiments showed that interactions of CHIKV HVD with CD2AP and FHL1 have additive effects on the efficiency of CHIKV replication. This study shows that CHIKV mutants with extensive modifications of FHL1- or both FHL1- and CD2AP-binding sites remain viable and develop spreading infection in multiple cell types. Our study also demonstrated that other members of the FHL family can bind to CHIKV HVD and thus may be involved in viral replication. IMPORTANCE Replication of chikungunya virus (CHIKV) is determined by a wide range of host factors. Previously, we have demonstrated that the hypervariable domain (HVD) of CHIKV nsP3 contains linear motifs that recruit defined families of host proteins into formation of functional viral replication complexes. Now, using NMR-based structural and biological approaches, we have characterized the binding site of the cellular FHL1 protein in CHIKV HVD and defined the CHK1-IN-2 biological significance of this interaction. In contrast to previously described binding of G3BP to CHIKV HVD, the FHL1-HVD interaction was found to not be a prerequisite of viral replication. However, the presence of FHL1 has a stimulatory effect on CHIKV infectivity and, subsequently, the infection spread. FHL1 and CD2AP proteins were found to have overlapping binding sites in CHIKV HVD and additive proviral functions. Elimination of the FHL1-binding site in the nsP3 HVD can be used for the development of stable, attenuated vaccine candidates. genus of the family (1). In natural circulation, CHIKV is transmitted by mosquito vectors between amplifying vertebrate hosts (2, 3). In mosquitoes, CHIKV develops persistent infection characterized by the high concentration of the virus in salivary glands. Upon infection by mosquito bites, vertebrate hosts develop acute febrile illness characterized by a high-level viremia, which is required for transmission to new mosquitoes during the blood meal. Humans can be infected by CHIKV by spillover from the enzootic transmission cycle. However, urban transmissions have also become common, and humans may serve as main amplifying hosts, with and being the transmission vectors (4). In humans, the CHIKV-induced disease is characterized by painful polyarthralgia, fever, and rash. In contrast to the Rabbit Polyclonal to OR52D1 diseases caused by most arthritogenic alphaviruses, CHIKV-induced arthralgia can last for months to years (5, 6). Thus, despite the fact that lethal outcomes are very rare, CHIKV represents an unquestionable public health threat. Previously, based on its geographical circulation, CHIKV was referred to as the Old World (OW) alphavirus. However, within the last 2 decades, it has demonstrated an unprecedented spread with wide occurrences in both the Old and New Worlds (7). As for other alphaviruses, the CHIKV genome (G RNA) is represented by a single-stranded RNA of positive polarity of 11.5?kb (8). After the release from infectious virions, G RNA serves as a template for translation of four nonstructural proteins, nsP1 to nsP4, which CHK1-IN-2 are the viral components of replication complexes (vRCs). Initially, nsPs are synthesized as polyprotein precursors P123 and P1234 (9). The partially processed products, P123 and nsP4, mediate the synthesis of the negative-strand RNA to form a double-stranded RNA (dsRNA) intermediate (10, 11). At later times postinfection, the completely processed nsPs function in the transcription of viral G RNA and subgenomic (SG) RNA (12, 13). The latter RNA functions as a template for translation of viral structural proteins, which ultimately package the newly synthesized viral genomes into infectious virions. vRCs reside in the membrane-bound organelles termed spherules (14). The mechanistical understanding of their assembly and function remains obscure. Their CHK1-IN-2 assembly requires participation of a large variety of host proteins, which are indispensable for viral replication. The sets of host factors in vRCs are specific to each alphavirus and particular cell type used in the experiments (15,C22). Nonstructural proteins nsP1, nsP2, and nsP4 have specific enzymatic functions required for the synthesis of viral RNAs and their posttranscriptional modifications (11). nsP3 is an exception, and to date, no direct functions in RNA synthesis have been ascribed to this protein. However, our previous studies and those of other research groups have demonstrated that nsP3 proteins of CHIKV and other alphaviruses are the key determinants of the recruitment of host factors to the sites of vRC assembly at the plasma membrane and to other cytoplasmic complexes, whose functions remain to be better understood (11, 16). Alphavirus nsP3 proteins contain two conserved structured domains (macro domain and alphavirus.
SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication