The Enterovirus 71 A-particle Forms a Gateway to Allow Genome Release: A CryoEM Study of Picornavirus Uncoating

Since its discovery in 1969, enterovirus 71 (EV71) has emerged as a serious worldwide health threat. This human pathogen of the picornavirus family causes hand, foot, and mouth disease, and also has the capacity to invade the central nervous system to cause severe disease and death. Upon binding to a host receptor on the cell surface, the virus begins a two-step uncoating process, first forming an expanded, altered "A-particle", which is primed for genome release. In a second step after endocytosis, an unknown trigger leads to RNA expulsion, generating an intact, empty capsid. Cryo-electron microscopy reconstructions of these two capsid states provide insight into the mechanics of genome release. The EV71 A-particle capsid interacts with the genome near the icosahedral two-fold axis of symmetry, which opens to the external environment via a channel ~10 Å in diameter that is lined with patches of negatively charged residues. After the EV71 genome has been released, the two-fold channel shrinks, though the overall capsid dimensions are conserved. These structural characteristics identify the two-fold channel as the site where a gateway forms and regulates the process of genome release. © 2013 Shingler et al

Purified Feline and Canine Transferrin Receptors Reveal Complex Interactions with the Capsids of Canine and Feline Parvoviruses That Correspond to Their Host Ranges

The cell infection processes and host ranges of canine parvovirus (CPV) and feline panleukopenia virus (FPV) are controlled by their capsid interactions with the transferrin receptors (TfR) on their host cells. Here, we expressed the ectodomains of wild-type and mutant TfR and tested those for binding to purified viral capsids and showed that different naturally variant strains of the viruses were associated with variant interactions with the receptors which likely reflect the optimization of the viral infection processes in the different hosts. While all viruses bound the feline TfR, reflecting their tissue culture host ranges, a naturally variant mutant of CPV (represented by the CPV type-2b strain) that became the dominant virus worldwide in 1979 showed significantly lower levels of binding to the feline TfR. The canine TfR ectodomain did not bind to a detectable level in the in vitro assays, but this appears to reflect the naturally low affinity of that interaction, as only low levels of binding were seen when the receptor was expressed on mammalian cells; however, that was sufficient to allow endocytosis and infection. The apical domain of the canine TfR controls the specific interaction with CPV capsids, as a canine TfR mutant altering a glycosylation site in that domain bound FPV, CPV-2, and CPV-2b capsids efficiently. Enzymatic removal of the N-linked glycans did not allow FPV binding to the canine TfR, suggesting that the protein sequence difference is itself important. The purified feline TfR inhibited FPV and CPV-2 binding and infection of feline cells but not CPV-2b, indicating that the receptor binding may be able to prevent the attachment to the same receptor on cells

Different mechanisms of antibody-mediated neutralization of parvoviruses revealed using the Fab fragments of monoclonal antibodies

Abstract Antibody binding and neutralization are major host defenses against viruses, yet the mechanisms are often not well understood. Eight monoclonal antibodies and their Fab fragments were tested for neutralization of canine parvovirus and feline panleukopenia virus. All IgGs neutralized >85% of virus infectivity. Two Fabs neutralized when present at 5 nM, while the others gave little or no neutralization even at 20-100 nM. The antibodies bind two antigenic sites on the capsids which overlap the binding site of the host transferrin receptor (TfR). There was no specific correlation between Fab binding affinity and neutralization. All Fabs reduced capsid binding of virus to purified feline TfR in vitro, but the highly neutralizing Fabs were more efficient competitors. All partially prevented binding and uptake of capsids by feline TfR on cells. The virus appears adapted to allow some infectivity in the presence of at least low levels of antibodies

Cryo-EM structure of catalytic ribonucleoprotein complex RNase MRP

Ribozyme-based RNase MRP is an essential eukaryotic enzyme involved in the maturation of rRNA and is evolutionarily related to RNase P. Here, the authors present the 3.0 Å cryo-EM structure of the S. cerevisiae RNase MRP holoenzyme, a 450 kDa ribonucleoprotein complex and compare it with RNase P

Microviridae, a Family Divided: Isolation, Characterization, and Genome Sequence of φMH2K, a Bacteriophage of the Obligate Intracellular Parasitic Bacterium Bdellovibrio bacteriovorus

A novel single-stranded DNA phage, φMH2K, of Bdellovibrio bacteriovorus was isolated, characterized, and sequenced. This phage is a member of the Microviridae, a family typified by bacteriophage φX174. Although B. bacteriovorus and Escherichia coli are both classified as proteobacteria, φMH2K is only distantly related to φX174. Instead, φMH2K exhibits an extremely close relationship to the Microviridae of Chlamydia in both genome organization and encoded proteins. Unlike the double-stranded DNA bacteriophages, for which a wide spectrum of diversity has been observed, the single-stranded icosahedral bacteriophages appear to fall into two distinct subfamilies. These observations suggest that the mechanisms driving single-stranded DNA bacteriophage evolution are inherently different from those driving the evolution of the double-stranded bacteriophages

Cryo EM Analysis Reveals Inherent Flexibility of Authentic Murine Papillomavirus Capsids

Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. However, studies have been hampered due to restricted tropism that makes production and purification of high titer virus problematic. This issue has been overcome by developing alternative HPV production methods such as virus-like particles (VLPs), which are devoid of a native viral genome. Structural studies have been limited in resolution due to the heterogeneity, fragility, and stability of the VLP capsids. The mouse papillomavirus (MmuPV1) presented here has provided the opportunity to study a native papillomavirus in the context of a common laboratory animal. Using cryo EM to solve the structure of MmuPV1, we achieved 3.3 Å resolution with a local symmetry refinement method that defined smaller, symmetry related subparticles. The resulting high-resolution structure allowed us to build the MmuPV1 asymmetric unit for the first time and identify putative L2 density. We also used our program ISECC to quantify capsid flexibility, which revealed that capsomers move as rigid bodies connected by flexible linkers. The MmuPV1 flexibility was comparable to that of a HPV VLP previously characterized. The resulting MmuPV1 structure is a promising step forward in the study of papillomavirus and will provide a framework for continuing biochemical, genetic, and biophysical research for papillomaviruses

Lifecycle of a human enterovirus.

<p>Three viral structural proteins (VP0, VP1, and VP3) function as a single structural subunit, the protomer. Five protomers then form a pentamer, twelve of which can self-associate to form a naturally occurring empty capsid (often referred to as procapsid). In pathway one, after the twelve pentamers form an empty procapsid (1A) the genome is inserted (1B). In the second proposed pathway 12 pentamers assemble around a genome to form a provirion (2). In this pathway pentamers that associate in the absence of genome would serve as a reservoir of capsid components in infected cells, and procapsid would be an off-pathway assembly byproduct. The provirion is a short-lived intermediate, as the autocatalytic cleavage of VP0 to form VP2 and VP4 is rapid and efficient. This cleavage results in the native virus, which upon binding to a cellular receptor transitions to an A-particle, which has expelled VP4 and exposed the N-terminus of VP1. An unknown secondary trigger causes the genome to egress, leaving behind an empty capsid shell. Note: The procapsid in this figure is shown in an expanded form, like that of EV71. Other picornavirus procapsids are the same diameter as their native virion (poliovirus and foot and mouth disease virus <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003240#ppat.1003240-Basavappa1" target="_blank">[20]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003240#ppat.1003240-Curry1" target="_blank">[21]</a>).</p

Cryo-electron microscopy image reconstruction data.

<p>Cryo-electron microscopy image reconstruction data.</p