Architecture of the chikungunya virus replication organelle

Abstract

Alphaviruses are mosquito-borne viruses that cause serious disease in humans and other mammals. Along with its mosquito vector, the Alphavirus chikungunya virus (CHIKV) has spread explosively in the last 20 years, and there is no approved treatment for chikungunya fever. On the plasma membrane of the infected cell, CHIKV generates dedicated organelles for viral RNA replication, so-called spherules. Whereas structures exist for several viral proteins that make up the spherule, the architecture of the full organelle is unknown. Here, we use cryo-electron tomography to image CHIKV spherules in their cellular context. This reveals that the viral protein nsP1 serves as a base for the assembly of a larger protein complex at the neck of the membrane bud. Biochemical assays show that the viral helicase-protease nsP2, while having no membrane affinity on its own, is recruited to membranes by nsP1. The tomograms further reveal that full-sized spherules contain a single copy of the viral genome in double-stranded form. Finally, we present a mathematical model that explains the membrane remodeling of the spherule in terms of the pressure exerted on the membrane by the polymerizing RNA, which provides a good agreement with the experimental data. The energy released by RNA polymerization is found to be sufficient to remodel the membrane to the characteristic spherule shape.

In situ characterization of late Chikungunya virus replication organelle

“…Surprisingly, CHIKV spherules imaged in the current study, whatever their localization on the PM, are heterogeneous in size, with a mean diameter of 90 nm, therefore contrasting with previous observations mainly performed from other model alphaviruses (i.e. SFV and SINV) or even for CHIKV for which ROs diameter was reported ranging from 50 to 70 nm at 6-8 hpi 48 , a time corresponding to the end of the first viral cycle. Analyzing more closely CHIKV ROs size distribution revealed at least three main categories.…”

“…The second ring of densities is supposed to correspond to nsP3 and associated cellular factors, including G3BP. Thus, the structural organization of RO neck revealed in our study is in agreement with that described recently from infected cells after 6-8 hpi 48,49 , meaning that the crown-like organization of the replication complex persists in ROs maintained at the PM even after 17 h of infection, arguing again for the functionality of ROs in our experimental conditions. Interestingly, the close inspection of ROs revealed that the crown organization of the complex is often difficult to see in individual sub-tomograms.…”

“…The variation of ROs diameters at the PM contrasts with homogeneity of spherule diameters in CPVs (50-60 nm). As the size of ROs in CPVs is very close to that of CHIKV ROs detected by others at the PM after 6-8 hpi 48,49 , this argues for a regulated internalization mechanism, appearing at the end of the replication cycle, it means in the 6-8h after CHIKV infection. Consequently, we suppose that during the first 6-8h of infection, the spherules replicate RNA, growing up to the optimal size of about 50-70 nm.…”

“…In a closer analysis of our data, considering the whole ROs population, we discerned several RO diameter maxima of 55, 64, 74.5, 84, 91, 100, 122, and 139 nm (Figure 3B). It is worth noting that some very recent cryo-EM studies reported RO with diameters of about 50 to 70 nm formed 6-8h after CHIKV infection 48,49 , a time point at which CHIKV first replication cycle is admitted to be complete, resulting in the presence of mature ROs containing fully-processed nsPs and dsRNA replication intermediates. Contrasting with this experimental scheme, our observations were set at 17h after the CHIKV challenge.…”

“…ROs with maximal diameter of 84-100 nm represent >60% of total events, while compartments with a size of 40-83 nm or 101-152 nm were more rarely detected. As the size of ROs was correlated with RNA genome length contained in these compartments 3,50 , and that the spherules of 50-70 nm were proposed to contained a single copy of the viral genome in double-stranded form 48 , it signifies that the vast majority of CHIKV ROs observed at the PM at 17 hpi, corresponds to ROs maintained at the PM after the first viral cycle, continuing to replicate and accumulate viral RNA in their lumen. The variation of ROs diameters at the PM contrasts with homogeneity of spherule diameters in CPVs (50-60 nm).…”