Phosphorylation Sites in the Hypervariable Domain in Chikungunya Virus nsP3 Are Crucial for Viral Replication

Abstract

Chikungunya virus (CHIKV, family Togaviridae) is a mosquito-transmitted alphavirus. The positive-sense RNA genome of CHIKV encodes four nonstructural proteins (nsP1–nsP4) that are virus-specific subunits of the RNA replicase. Among nsP functions, those of nsP3 are least understood. The C-terminal hypervariable domain (HVD) in nsP3 is disordered and serves as a platform for interactions with multiple host proteins. For Sindbis virus (SINV) and Semliki Forest virus (SFV), the nsP3 HVD has been shown to be phosphorylated. Deletion of phosphorylated regions has a mild effect on the growth of SFV and SINV in vertebrate cells. Using radiolabeling, we demonstrated that nsP3 in CHIKV and o’nyong-nyong virus is also phosphorylated. We showed that the phosphorylated residues in CHIKV nsP3 are not clustered at the beginning of the HVD. The substitution of 20 Ser/Thr residues located in the N-terminal half of the HVD or 26 Ser/Thr residues located in its C-terminal half with Ala residues reduced the activity of the CHIKV replicase and the infectivity of CHIKV in mammalian cells. Furthermore, the substitution of all 46 potentially phosphorylated residues resulted in the complete loss of viral RNA synthesis and infectivity. The mutations did not affect the interaction of the HVD in nsP3 with the host G3BP1 protein; interactions with CD2AP, BIN1 and FHL1 proteins were significantly reduced, but not abolished. Thus, CHIKV differs from SFV and SINV both in the location of the phosphorylated residues in the HVD in nsP3 and, significantly, in their effect on replicase activity and virus infectivity. IMPORTANCE: Chikungunya virus (CHIKV) outbreaks have affected millions of people, creating a need for the development of antiviral approaches. Nonstructural protein 3 (nsP3) is a component of the CHIKV RNA replicase and is involved in interactions with host proteins and signaling cascades. Phosphorylation of the hypervariable domain (HVD) in nsP3 is important for the virulent alphavirus phenotype. Here, we demonstrate that nsP3 in CHIKV is phosphorylated and that the phosphorylation sites in the HVD are distributed in a unique pattern. Furthermore, the abrogation of some of the phosphorylation sites results in the attenuation of CHIKV, while abolishing all the phosphorylation sites completely blocked its replicase activity. Thus, the phosphorylation of nsP3 and/or the phosphorylation sites in nsP3 have a major impact on CHIKV infectivity. Therefore, they represent promising targets for antiviral compounds and CHIKV attenuation. In addition, this new information offers valuable insight into the vast network of virus-host interactions.

NAP1L1 and NAP1L4 binding to Hypervariable Domain of Chikungunya Virus nsP3 Protein is bivalent and requires phosphorylation

“…Importantly, it has been demonstrated for Mayaro virus that inhibition of CK2 could also strongly reduce viral replication (54). The requirement for HVD phosphorylation for interaction with other host partners is not so obvious, but might also result in higher affinities of protein binding, and this provides a plausible explanation for the HVD phosphorylation that was described for all known alphaviruses (11,13,(34)(35)(36)(37)(38)(39)(40)(41). Taken together, the accumulating data strongly suggest an additional level of the viral life cycle regulation by phosphorylation of HVD, which leads to differential interactions with host proteins.…”

“…1 and 2). However, alphavirus HVDs are known to be highly phosphorylated (11,13,(34)(35)(36)(37)(38)(39)(40)(41), and proteins produced in E.coli lack this post translational modification. Moreover, it was previously suggested, that NAP1L1 interaction with HCV NS5 protein is dependent on phosphorylation (42).…”

Crystal structures of alphavirus nonstructural protein 4 (nsP4) reveal an intrinsically dynamic RNA-dependent RNA polymerase fold

“…Having determined the crystal structure of RRV RdRp, we next analyzed the functional importance of several of its key structural features. As the introduction of unfavorable mutations into an infectious clone tends to result in no data (lethal phenotype) or in the rapid appearance of revertants or compensating mutations (30,31), we instead used a novel two-component transreplication system (Figure 5a), which has extremely high sensitivity (Lello et al, unpublished manuscript). Briefly, the system is based on the ability of separately expressed P123 and nsP4 of alphaviruses to form a functional RNA replicase.…”

The Putative Roles and Functions of Indel, Repetition and Duplication Events in Alphavirus Non-Structural Protein 3 Hypervariable Domain (nsP3 HVD) in Evolution, Viability and Re-Emergence

“…Interestingly, nsP3 is the only protein which can be phosphorylated among all alphavirus replicase proteins. The nsP3 HVD is heavily phosphorylated and is involved in signaling cascades, alphavirus RC formation, and alphavirus virulent phenotypes [176,177]. Structural studies on nsP3 HVD are key to gaining insight into alphavirus RC assembly and functions during viral replication at the molecular level.…”

“…The nsP3 HVD functions as a hub allowing interaction of various host proteins [177]. Despite being highly variable and having non-conserved genetic characteristics, nsP3 HVD from different species of alphavirus still consist of several conserved identified linear motifs that have been proven to interact with specific sets of host cellular factors from at least three protein families.…”