Virus Assembly Pathways: Straying Away but Not Too Far

Abstract

viruses, the CPs can self-assemble in vitro around a variety of non-genomic cargo, [6] forming shells that can be structurally identical to those of the wild type (wt) virus. However, wt virus assembly inside the heterogeneous, crowded environment of a host cell cytoplasm surprisingly leads to the large majority of virions being laden with genomic RNA. [7] The reason for non-specific encapsulation in vitro is relatively well understood. [5,8] The main driving force behind assembly at physiological ionic strengths are electrostatic interactions. [9] By comparison, the rate of empty capsids assembly, which can occur when electrostatic interactions are screened, is several orders of magnitude slower than co-assembly of coat proteins in presence of polyanionic species. [10] The question is then, how does a virus avoid production of virus-like particles that encapsulate many of the smaller, non-viral, transient RNAs and other polyions occurring in the cytoplasm? In attempting to answer this long-standing question we have studied the nature of chimeras which assemble out of small ssDNA oligonucleotides and viral coat protein. Cryo-electron microscopy (cryo-EM) and charge detection mass spectrometry (CDMS) analysis of in vitro assembly products suggest that CP shells do readily form around multiple oligonucleotides. However, these shells have specific, strained structures which easily split into large fragments. These may provide intermediates for correct, fast virion growth when cognate RNA, containing appropriate packaging signals becomes available. [11-13] 2. Results and Discussion In this work, virus-like particles (VLPs) were formed by mixing purified coat proteins of the brome mosaic virus (BMV) with two types of ssDNA oligonucleotides, both 52 nucleotides long. The two oligonucleotide fragments had different tertiary structures: the first one, hereafter called oligoB, originated from the BMV RNA genome sequence that interacts with the BMV CP N-terminal arm. [14,15] The second oligomer was a linear polyA polymer with no tertiary structure (see Figures S1 and S2, Supporting Information for assembly conditions, biochemical characterization, and transmission electron microscopy (TEM) characterization). To determine the masses of the VLPs formed, we employed charge detection mass spectrometry (CDMS). CDMS measures Non-enveloped RNA viruses pervade all domains of life. In a cell, they coassemble from viral RNA and capsid proteins. Virus-like particles can form in vitro where virtually any non-cognate polyanionic cargo can be packaged. How only viral RNA gets selected for packaging in vivo, in presence of myriad other polyanionic species, has been a puzzle. Through a combination of charge detection mass spectrometry and cryo-electron microscopy, it is determined that co-assembling brome mosaic virus (BMV) coat proteins and nucleic acid oligomers results in capsid structures and stoichiometries that differ from the icosahedral virion. These previously unknown shell structures are strained and less stable than the native ...

Cryo-EM reconstructions of BMV-derived virus-like particles reveal assembly defects in the icosahedral lattice structure

“…This suggests that such shells composed only of pentamers are not formed by the full-length BMV CP but only when the 35 N-terminal residues are truncated. 4,26 We also did not observe multiplets (a few protein shells joined together), which confirms that such VLP species are formed only in the presence of a long polyelectrolyte. 4,10,22 Analyzing the topography of both meVLP types, we observed that in each type, one side of the protein shell seems to be better ordered and complete than the opposite side (Fig.…”

“…However, we detected H12 shells but not H8 shells as Bond et al did. 4 Most probably, H12 is stable enough in the absence of the polyelectrolyte, while the stabilization of the H8 shell requires a polyanion similar to ssDNA that can interact with CP through electrostatic interactions.…”

“…1,2 The increasing interest in VLP application has been reflected by the growing number of studies on their assembly and disassembly processes as well as on their cellular internalization, cargo capacity, or modifications of their external surface in order to direct them to target cells. [3][4][5][6][7] Recently, VLPs composed of the native capsid proteins from plant viruses belonging to the bromovirus genus, i.e. brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV), have been shown to easily internalize fluorophores into breast tumor cells.…”

RNA Structures and Their Role in Selective Genome Packaging

“…In the absence of genomic RNA, retroviruses package cellular RNAs, and retroviral cores can be disrupted upon treatment with RNase [ 35 ]. Moreover, recent work with the non-enveloped brome mosaic virus (BMV) has shown that when capsid subunits assemble on small non-viral RNAs it forms unstable shells with non-icosahedral structures that may be rapidly recycled into icosahedral capsids when the genomic RNA becomes available [ 36 ]. Furthermore, impressive cryo-electron microscopy (cryo-EM) reconstruction of the MS2 bacteriophage demonstrate how distinct, but non-uniformly distributed RNA structures favour assembly of the correct icosahedral capsid structure over non-productive octahedral structures [ 16 ].…”

“…Furthermore, impressive cryo-electron microscopy (cryo-EM) reconstruction of the MS2 bacteriophage demonstrate how distinct, but non-uniformly distributed RNA structures favour assembly of the correct icosahedral capsid structure over non-productive octahedral structures [ 16 ]. Evidently, optimal viral assembly depends on the biophysical properties of the genomic RNA, which in turn is driven by a myriad of factors, including RNA sequence, structure [ 37 , 38 ] and size [ 36 ].…”