Partially Uncleaved Alphavirus Replicase Forms Spherule Structures in the Presence and Absence of RNA Template

Abstract

Alphaviruses are positive-strand RNA viruses expressing their replicase as a polyprotein, P1234, which is cleaved to four final products, nonstructural proteins nsP1 to nsP4. The replicase proteins together with viral RNA and host factors form membrane invaginations termed spherules, which act as the replication complexes producing progeny RNAs. We have previously shown that the wild-type alphavirus replicase requires a functional RNA template and active polymerase to generate spherule structures. However, we now find that specific partially processed forms of the replicase proteins alone can give rise to membrane invaginations in the absence of RNA or replication. The minimal requirement for spherule formation was the expression of properly cleaved nsP4, together with either uncleaved P123 or with the combination of nsP1 and uncleaved P23. These inactive spherules were morphologically less regular than replication-induced spherules. In the presence of template, nsP1 plus uncleaved P23 plus nsP4 could efficiently assemble active replication spherules producing both negative-sense and positive-sense RNA strands. P23 alone did not have membrane affinity, but could be recruited to membrane sites in the presence of nsP1 and nsP4. These results define the set of viral components required for alphavirus replication complex assembly and suggest the possibility that it could be reconstituted from separately expressed nonstructural proteins. All positive-strand RNA viruses extensively modify host cell membranes to serve as efficient platforms for viral RNA replication. Alphaviruses and several other groups induce protective membrane invaginations (spherules) as their genome factories. Most positive-strand viruses produce their replicase as a polyprotein precursor, which is further processed through precise and regulated cleavages. We show here that specific cleavage intermediates of the alphavirus replicase can give rise to spherule structures in the absence of viral RNA. In the presence of template RNA, the same intermediates yield active replication complexes. Thus, partially cleaved replicase proteins play key roles that connect replication complex assembly, membrane deformation, and the different stages of RNA synthesis.

Deciphering the Nucleotide and RNA Binding Selectivity of the Mayaro Virus Macro Domain

“…Note that signals of the neighboring residues Gly30-Asp31, Cys34-Ala36, Ala38, Gly112-Phe114 and Asp119-Arg120 are broadened beyond detection in the 1 H, 15 N heteronuclear single quantum coherence (HSQC) spectrum of the apo form of MAYV MD. Thus, their 1 H, 15 N correlation peaks could not be assigned, strongly suggesting that the loops upstream to helices α1 and α3 undergo conformational exchange processes that may be relevant for the selectivity of substrate binding [52]. These data support the plasticity of two functionally important loops ( Fig.…”

“…However, the residues exhibiting CSPs are practically the same as in the case of ADP. Similarly, the 1 H, 15 N HSQC cross peaks of Ile11, Ala23, Asn24, Gly32, Val33, Val69, Leu108, Ser110 and Ala116 remained broadened beyond detection even after the last addition of ATP ( Fig. 2f).…”

“…In addition, adjacent amino acids like Ala9, Ala12, Thr13, Gly27, Val29, Asn72, Phe73, Leu109, Ser115 and G117 were also affected exhibiting a slow-exchange process during the interaction. The backbone HN resonances of Ile11, His25, Val37, Phe45, Ala68 and Lys146 broadened beyond detection, while those of Ala22, Gly30, Asp31, Cys34, Arg35, Ala36, Ala38, Gly112, Cys143 and Arg144, which were unassigned in the spectra of the apo MAYV MD, could be assigned in the 1 H, 15 N HSQC spectrum of the MAYV MD-ADP-r complex. These data suggest that the majority of the residues, which participate in ligand recognition and binding, are located near the positively charged ligand-binding cleft ( Figs.…”

“…A series of adenosine-based molecules was used to explore the ability of MAYV MD to bind adenosine derivatives as well as to elucidate the impact of individual chemical moieties on the MAYV MD-ligand interaction and the role of certain residues in ligand recognition. Titrations of MAYV MD with ADP-r, ADP, adenosine triphosphate (ATP), adenosine monophosphate (AMP) and NAD+ were monitored by acquisition of 1 H, 15 N HSQC spectra and showed chemical exchange between free and ligand-bound MD ranging from fast to slow on the NMR timescale.…”

“…The replication of Alphavirus occurs in spherules [13,14] containing four non-structural proteins (nsP1 to nsP4) [15] resulting from processing of the precursors P123 and P1234. NsP1 is carrying the enzymatic activities for the mRNA cap formation, nsP2 has a N-terminal domain with multiple enzymatic activities (e.g., ATPase, RNA helicase, RNA triphosphatase) [16][17][18].…”