|Journal of Virology Structure and Assembly|
In order to initiate an infection, viruses need to deliver their genomes into cells. This involves uncoating the genome and transporting it to the cytoplasm. The process of genome delivery is not well understood for nonenveloped viruses. We address this gap in our current knowledge by studying the uncoating of the nonenveloped human cardiovirus Saffold virus 3 (SAFV-3) of the family Picornaviridae. SAFVs cause diseases ranging from gastrointestinal disorders to meningitis. We present a structure of a native SAFV-3 virion determined to 2.5 AAring; by X-ray crystallography and an 11-AAring;-resolution cryo-electron microscopy reconstruction of an "altered" particle that is primed for genome release. The altered particles are expanded relative to the native virus and contain pores in the capsid that might serve as channels for the release of VP4 subunits, N termini of VP1, and the RNA genome. Unlike in the related enteroviruses, pores in SAFV-3 are located roughly between the icosahedral 3- and 5-fold axes at an interface formed by two VP1 and one VP3 subunit. Furthermore, in native conditions many cardioviruses contain a disulfide bond formed by cysteines that are separated by just one residue. The disulfide bond is located in a surface loop of VP3. We determined the structure of the SAFV-3 virion in which the disulfide bonds are reduced. Disruption of the bond had minimal effect on the structure of the loop, but it increased the stability and decreased the infectivity of the virus. Therefore, compounds specifically disrupting or binding to the disulfide bond might limit SAFV infection.
IMPORTANCE A capsid assembled from viral proteins protects the virus genome during transmission from one cell to another. However, when a virus enters a cell the virus genome has to be released from the capsid in order to initiate infection. This process is not well understood for nonenveloped viruses. We address this gap in our current knowledge by studying the genome release of Human Saffold virus 3. Saffold viruses cause diseases ranging from gastrointestinal disorders to meningitis. We show that before the genome is released, the Saffold virus 3 particle expands, and holes form in the previously compact capsid. These holes serve as channels for the release of the genome and small capsid proteins VP4 that in related enteroviruses facilitate subsequent transport of the virus genome into the cell cytoplasm.
Paramyxoviridae consist of a large family of enveloped, negative-sense, nonsegmented single-stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN [also called H or G depending on virus type and substrate]), which acts as a receptor binding protein, and a fusion (F) protein, which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN 4-helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross-linking as well as functional assays to investigate the role of the structurally unresolved membrane-proximal stalk region (MPSR) (residues 37 to 58) of HN in the context of headless and full-length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F triggering.
IMPORTANCE Paramyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F), to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane-anchored flexible stalk of ~80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane-proximal stalk region (MPSR) (HN, residues 37 to 56) of the paramyxovirus parainfluenza virus (PIV5), a region of the HN stalk that has not had its structure determined by X-ray crystallography. Our data suggest that the MPSR influences receptor binding and neuraminidase activity via an indirect mechanism. Moreover, the receptor binding head group stabilizes the 4HB stalk as part of the general mechanism to fine-tune F-activation.
Although the X174 H protein is monomeric during procapsid morphogenesis, 10 proteins oligomerize to form a DNA translocating conduit (H-tube) for penetration. However, the timing and location of H-tube formation are unknown. The H-tube's highly repetitive primary and quaternary structures made it amenable to a genetic analysis using in-frame insertions and deletions. Length-altered proteins were characterized for the ability to perform the protein's three known functions: participation in particle assembly, genome translocation, and stimulation of viral protein synthesis. Insertion mutants were viable. Theoretically, these proteins would produce an assembled tube exceeding the capsid's internal diameter, suggesting that virions do not contain a fully assembled tube. Lengthened proteins were also used to test the biological significance of the crystal structure. Particles containing H proteins of two different lengths were significantly less infectious than both parents, indicating an inability to pilot DNA. Shortened H proteins were not fully functional. Although they could still stimulate viral protein synthesis, they either were not incorporated into virions or, if incorporated, failed to pilot the genome. Mutant proteins that failed to incorporate contained deletions within an 85-amino-acid segment, suggesting the existence of an incorporation domain. The revertants of shortened H protein mutants fell into two classes. The first class duplicated sequences neighboring the deletion, restoring wild-type length but not wild-type sequence. The second class suppressed an incorporation defect, allowing the use of the shortened protein.
IMPORTANCE The H-tube crystal structure represents the first high-resolution structure of a virally encoded DNA-translocating conduit. It has similarities with other viral proteins through which DNA must travel, such as the aalpha;-helical barrel domains of P22 portal proteins and T7 proteins that form tail tube extensions during infection. Thus, the H protein serves as a paradigm for the assembly and function of long aalpha;-helical supramolecular structures and nanotubes. Highly repetitive in primary and quaternary structure, they are amenable to structure-function analyses using in-frame insertions and deletions as presented herein.