Structural studies on the authentic mumps virus nucleocapsid showing uncoiling by the phosphoprotein.

Mumps virus (MuV) is a highly contagious pathogen, and despite extensive vaccination campaigns, outbreaks continue to occur worldwide. The virus has a negative-sense, single-stranded RNA genome that is encapsidated by the nucleocapsid protein (N) to form the nucleocapsid (NC). NC serves as the template for both transcription and replication. In this paper we solved an 18-Å-resolution structure of the authentic MuV NC using cryo-electron microscopy. We also observed the effects of phosphoprotein (P) binding on the MuV NC structure. The N-terminal domain of P (PNTD) has been shown to bind NC and appeared to induce uncoiling of the helical NC. Additionally, we solved a 25-Å-resolution structure of the authentic MuV NC bound with the C-terminal domain of P (PCTD). The location of the encapsidated viral genomic RNA was defined by modeling crystal structures of homologous negative strand RNA virus Ns in NC. Both the N-terminal and C-terminal domains of MuV P bind NC to participate in access to the genomic RNA by the viral RNA-dependent-RNA polymerase. These results provide critical insights on the structure-function of the MuV NC and the structural alterations that occur through its interactions with P.

Cryo-EM analysis of the organization of BclA and BxpB in the Bacillus anthracis exosporium.

Bacillus anthracis and other pathogenic Bacillus species form spores that are surrounded by an exosporium, a balloon-like layer that acts as the outer permeability barrier of the spore and contributes to spore survival and virulence. The exosporium consists of a hair-like nap and a paracrystalline basal layer. The filaments of the nap are comprised of trimers of the collagen-like glycoprotein BclA, while the basal layer contains approximately 20 different proteins. One of these proteins, BxpB, forms tight complexes with BclA and is required for attachment of essentially all BclA filaments to the basal layer. Another basal layer protein, ExsB, is required for the stable attachment of the exosporium to the spore. To determine the organization of BclA and BxpB within the exosporium, we used cryo-electron microscopy, cryo-sectioning and crystallographic analysis of negatively stained exosporium fragments to compare wildtype spores and mutant spores lacking BclA, BxpB or ExsB (ΔbclA, ΔbxpB and ΔexsB spores, respectively). The trimeric BclA filaments are attached to basal layer surface protrusions that appear to be trimers of BxpB. The protrusions interact with a crystalline layer of hexagonal subunits formed by other basal layer proteins. Although ΔbxpB spores retain the hexagonal subunits, the basal layer is not organized with crystalline order and lacks basal layer protrusions and most BclA filaments, indicating a central role for BxpB in exosporium organization.

Structure and size determination of bacteriophage P2 and P4 procapsids: function of size responsiveness mutations.

Bacteriophage P4 is dependent on structural proteins supplied by a helper phage, P2, to assemble infectious virions. Bacteriophage P2 normally forms an icosahedral capsid with T=7 symmetry from the gpN capsid protein, the gpO scaffolding protein and the gpQ portal protein. In the presence of P4, however, the same structural proteins are assembled into a smaller capsid with T=4 symmetry. This size determination is effected by the P4-encoded protein Sid, which forms an external scaffold around the small P4 procapsids. Size responsiveness (sir) mutants in gpN fail to assemble small capsids even in the presence of Sid. We have produced large and small procapsids by co-expression of gpN with gpO and Sid, respectively, and applied cryo-electron microscopy and three-dimensional reconstruction methods to visualize these procapsids. gpN has an HK97-like fold and interacts with Sid in an exposed loop where the sir mutations are clustered. The T=7 lattice of P2 has dextro handedness, unlike the laevo lattices of other phages with this fold observed so far.

Structure of the Capsid Size-Determining Scaffold of "Satellite" Bacteriophage P4.

P4 is a mobile genetic element (MGE) that can exist as a plasmid or integrated into its Escherichia coli host genome, but becomes packaged into phage particles by a helper bacteriophage, such as P2. P4 is the original example of what we have termed "molecular piracy", the process by which one MGE usurps the life cycle of another for its own propagation. The P2 helper provides most of the structural gene products for assembly of the P4 virion. However, when P4 is mobilized by P2, the resulting capsids are smaller than those normally formed by P2 alone. The P4-encoded protein responsible for this size change is called Sid, which forms an external scaffolding cage around the P4 procapsids. We have determined the high-resolution structure of P4 procapsids, allowing us to build an atomic model for Sid as well as the gpN capsid protein. Sixty copies of Sid form an intertwined dodecahedral cage around the T = 4 procapsid, making contact with only one out of the four symmetrically non-equivalent copies of gpN. Our structure provides a basis for understanding the sir mutants in gpN that prevent small capsid formation, as well as the nms "super-sid" mutations that counteract the effect of the sir mutations, and suggests a model for capsid size redirection by Sid.

Codon usage optimization of HIV type 1 subtype C gag, pol, env, and nef genes: in vitro expression and immune responses in DNA-vaccinated mice.

Codon usage optimization of human immunodeficiency virus type 1 (HIV-1) structural genes has been shown to increase protein expression in vitro as well as in the context of DNA vaccines in vivo; however, all optimized genes reported thus far are derived from HIV-1 (group M) subtype B viruses. Here, we report the generation and biological characterization of codon usage-optimized gag, pol, env (gp160, gp140, gp120), and nef genes from a primary (nonrecombinant) HIV-1 subtype C isolate. After transfection into 293T cells, optimized subtype C genes expressed one to two orders of magnitude more protein (as determined by immunoblot densitometry) than the corresponding wild-type constructs. This effect was most pronounced for gp160, gp140, Gag, and Pol (>250-fold), but was also observed for gp120 and Nef (45- and 20-fold, respectively). Optimized gp160- and gp140-derived glycoproteins were processed, incorporated into virus particles, and mediated virus entry when expressed in trans to complement an env-minus HIV-1 provirus. Mice immunized with optimized gp140 DNA developed antibody as well as CD4+ and CD8+ T cell immune responses that were orders of magnitude greater than those of mice immunized with wild-type gp140 DNA. These data confirm and extend previous studies of codon usage optimization of HIV-1 genes to the most prevalent group M subtype. Our panel of matched optimized and wild-type subtype C genes should prove valuable for studies of protein expression and function, the generation of subtype-specific immunological reagents, and the production of DNA-based sub-unit vaccines directed against a broader spectrum of viruses.

Assembly of bacteriophage 80α capsids in a Staphylococcus aureus expression system.

80α is a temperate, double-stranded DNA bacteriophage of Staphylococcus aureus that can act as a "helper" for the mobilization of S. aureus pathogenicity islands (SaPIs), including SaPI1. When SaPI1 is mobilized by 80α, the SaPI genomes are packaged into capsids that are composed of phage proteins, but that are smaller than those normally formed by the phage. This size determination is dependent on SaPI1 proteins CpmA and CpmB. Here, we show that co-expression of the 80α capsid and scaffolding proteins in S. aureus, but not in E. coli, leads to the formation of procapsid-related structures, suggesting that a host co-factor is required for assembly. The capsid and scaffolding proteins also undergo normal N-terminal processing upon expression in S. aureus, implicating a host protease. We also find that SaPI1 proteins CpmA and CpmB promote the formation of small capsids upon co-expression with 80α capsid and scaffolding proteins in S. aureus.

Functional domains of the bacteriophage P2 scaffolding protein: identification of residues involved in assembly and protease activity.

Bacteriophage P2 encodes a scaffolding protein, gpO, which is required for correct assembly of P2 procapsids from the gpN major capsid protein. The 284 residue gpO protein also acts as a protease, cleaving itself into an N-terminal fragment, O, that remains in the capsid following maturation. In addition, gpO is presumed to act as the maturation protease for gpN, which is N-terminally processed to N, accompanied by DNA packaging and capsid expansion. The protease activity of gpO resides in the N-terminal half of the protein. We show that gpO is a classical serine protease, with a catalytic triad comprised of Asp 19, His 48 and Ser 107. The C-terminal 90 amino acids of gpO are required and sufficient for capsid assembly. This fragment contains a predicted alpha-helical segment between residues 197 and 257 and exists as a multimer in solution, suggesting that oligomerization is required for scaffolding activity. Correct assembly requires the C-terminal cysteine residue, which is most likely involved in transient gpN interactions. Our results suggest a model for gpO scaffolding action in which the N-terminal half of gpO binds strongly to gpN, while oligomerization of the C-terminal alpha-helical domain of gpO and transient interactions between Cys 284 and gpN lead to capsid assembly.