Removal of Divalent Cations Induces Structural Transitions in Red Clover Necrotic Mosaic Virus , Revealing a Potential Mechanism for RNA Release

Abstract

The structure of Red clover necrotic mosaic virus (RCNMV), an icosahedral plant virus, was resolved to 8.5 Å by cryoelectron microscopy. The virion capsid has prominent surface protrusions and subunits with a clearly defined shell and protruding domains. The structures of both the individual capsid protein (CP) subunits and the entire virion capsid are consistent with other species in the Tombusviridae family. Within the RCNMV capsid, there is a clearly defined inner cage formed by complexes of genomic RNA and the amino termini of CP subunits. An RCNMV virion has approximately 390 ؎ 30 Ca 2؉ ions bound to the capsid and 420 ؎ 25 Mg 2؉ions thought to be in the interior of the capsid. Depletion of both Ca 2؉ and Mg 2؉ ions from RCNMV leads to significant structural changes, including (i) formation of 11-to 13-Å-diameter channels that extend through the capsid and (ii) significant reorganization within the interior of the capsid. Genomic RNA within native capsids containing both Ca 2؉ and Mg 2؉ ions is extremely resistant to nucleases, but depletion of both of these cations results in nuclease sensitivity, as measured by a significant reduction in RCNMV infectivity. These results indicate that divalent cations play a central role in capsid dynamics and suggest a mechanism for the release of viral RNA in low-divalent-cation environments such as those found within the cytoplasm of a cell.The Tombusviridae family consists of small, icosahedral plant viruses that are transmitted through the soil and infect their hosts via the root system (34). Given that these viruses must survive harsh environments, it is not surprising that they possess unusually stable and robust capsids. This high degree of virion stability raises the question of how the intracellular environment triggers disassembly or minimally exposes the genome for translation. Structural analyses of several plant viruses have revealed that maintenance of a stable capsid conformation is dependent on the presence of divalent cations bound to the capsid (25,33,36). It has been a longstanding hypothesis that swelling and other conformational changes induced by ion extraction from virions are critical for a productive viral life cycle (13). For Tomato bushy stunt virus (TBSV), the type species of the genus Tombusvirus within the Tombusviridae family, it was hypothesized that Ca 2ϩ ions would be released from viral capsids within infected cells, leading to virion swelling sufficient to expose the viral genome.Crystallographic studies of TBSV (28) revealed a capsid formed by 180 chemically identical capsid protein (CP) subunits in three quasiequivalent conformations (A, B, and C). Each CP subunit is composed of three distinct structural domains, which include the RNA-interacting (R), shell (S), and protruding (P) domains. The conformational differences that distinguish the A, B, and C subunits are localized within the hinge regions between the respective S and P domains. These hinges point either down (in A-B dimers) or up (in C-C dimers). In addition, the loop that ...

Chimeric viruses blur the borders between the major groups of eukaryotic single-stranded DNA viruses

“…S1). Similar pattern of conservation has been also observed in tombusviruses 43 . Closer examination of the multiple alignment of CHIV, tombusviral and oomycete-infecting virus (SmV-A and PhV-A) CP sequences shows that CHIV CPs are more closely related to the proteins of SmV-A and PhV-A than they are to the CPs of tombusviruses.…”

A Mathematical Approach to Structural Transitions in Viral Capsids

“…Examples include the maturation of the viral capsid from a procapsid into an expanded form in HK97, or the opening of pores in CCMV, TBSV or RCNMV, through which the genomic material is ejected. 24,26,27 Following Indelicato et al, 11 we demonstrate that such structural transitions can be understood via a crystallographic approach as illustrated in Fig. 1.…”

Nanotechnology-based approaches in anticancer research

“…Moreover, VLPs can be modified with polymers such as PEG to improve their half-life in the host by moderating their immunogenicity. 43 Different viruses such as cowpea chlorotic mottle virus (CCMV), a member of the bromovirus group of the Bromoviridae, 43 cowpea mosaic virus, and red clover necrotic mosaic virus 88 have been reported as viral nanocarriers. Recently, nanocarriers based on CCMV have also been suggested.…”

“…84,87 virus-mediated nanocarriers for drug delivery Different virus-based nanocarriers for drug delivery have also been reported in scientific literature as an emerging nanocarrier platform. 43,[88][89][90] The versatile hierarchical assembly of viral coat protein subunits provides a natural and easy way of drug packaging. Virus-like particles (VLPs) can easily meet the requirements needed for a drug nanocarrier system, such as biocompatibility, water solubility, and high uptake efficiency.…”