Purification of Highly Active Alphavirus Replication Complexes Demonstrates Altered Fractionation of Multiple Cellular Membranes

Abstract

Positive-strand RNA viruses replicate their genomes in membrane-associated structures; alphaviruses and many other groups induce membrane invaginations called spherules. Here, we established a protocol to purify these membranous replication complexes (RCs) from cells infected with Semliki Forest virus (SFV). We isolated SFV spherules located on the plasma membrane and further purified them using two consecutive density gradients. This revealed that SFV infection strongly modifies cellular membranes. We removed soluble proteins, the Golgi and most of the mitochondria, but plasma membrane, endoplasmic reticulum (ER) and late endosome markers enriched in the membrane fraction that contained viral RNA synthesizing activity, replicase proteins and minus- and plus-strand RNA. Electron microscopy revealed that the purified membranes displayed spherule-like structures with a narrow neck. This membrane enrichment was specific to viral replication as such a distribution of membrane markers was only observed after infection. Besides the plasma membrane, SFV infection remodeled the ER, and the co-fractionation of the RC-carrying plasma membrane and ER suggests that SFV may recruit ER proteins or membrane to the site of replication. The purified RCs were highly active in synthesizing both genomic and subgenomic RNA. Detergent solubilization destroyed the replication activity demonstrating that the membrane association of the complex is essential. Most of the newly made RNA was in double-stranded replicative molecules but the purified complexes also produced single-stranded RNA as well as released newly made RNA. This indicates that the purification established here maintained the functionality of RCs and thus enables further structural and functional studies of active RCs. Similar to all positive-strand RNA viruses, the arthropod-borne alphaviruses induce membranous genome factories but little is known about the arrangement of viral replicase proteins and the presence of host proteins in these replication complexes. To improve our knowledge of alphavirus RNA-synthesizing complexes, we isolated and purified them from infected mammalian cells. Detection of viral RNA and replication assays revealed that these complexes are abundant and highly active when located on the plasma membrane. After multiple purification steps, they remain functional in synthesizing and releasing viral RNA. Besides the plasma membrane, markers for the endoplasmic reticulum and late endosomes enriched with the replication complexes demonstrating that alphavirus infection modified cellular membranes beyond inducing replication spherules on the plasma membrane. We have here developed a gentle purification method to obtain large quantities of highly active replication complexes, and similar methods can be applied to other positive-strand RNA viruses.

Separate domains of G3BP promote efficient clustering of alphavirus replication complexes and recruitment of the translation initiation machinery

“…Our work is consistent with that model and shows that the molecular link between the viral CPVs and the cellular translation apparatus is G3BP1, recruited by nsP3. Other, more recent work has provided evidence that the newly produced alphaviral RNAs, upon exit from the spherule neck are protected from degradation by cellular RNases [46,47]. Indeed, a role for G3BP in protection of new viral genomic RNAs has been proposed [16], mediated by either or both of the RRM and RGG domains.…”

The RNA Capping Enzyme Domain in Protein A is Essential for Flock House Virus Replication

“…This system yields high levels of FHV RNA synthesis and amplification, exceeding the level of alphavirus RNA, and prominent spherules on mitochondrial surfaces ( Figure 2 and Figure 6 ). The system can be used as a basis for in vitro replication experiments, in which, similar to alphaviruses, mainly positive strand RNAs are produced ( Figure 3 ) [ 34 , 49 ]. In contrast to a recent study [ 36 ], we find that the endogenous template present inside the cells is sufficient for replication activity in vitro.…”

The Alphavirus Exit Pathway: What We Know and What We Wish We Knew

“…Through an intricate series of steps, the nsPs assemble a replication complex to produce more copies of the gRNA through a negative strand RNA intermediate. Viral RNA synthesis occurs within membrane spherules located on the plasma membrane and on cytopathic vacuole type I (CPVI) structures [ 23 , 24 , 25 ]. CPVI are membranous replication structures induced during viral infection.…”