Specific inter-domain interactions stabilize a compact HIV-1 Gag conformation.

HIV-1 Gag is a large multidomain poly-protein with flexible unstructured linkers connecting its globular subdomains. It is compact when in solution but assumes an extended conformation when assembled within the immature HIV-1 virion. Here, we use molecular dynamics (MD) simulations to quantitatively characterize the intra-domain interactions of HIV-1 Gag. We find that the matrix (MA) domain and the C-terminal subdomain CActd of the CA capsid domain can form a bound state. The bound state, which is held together primarily by interactions between complementary charged and polar residues, stabilizes the compact state of HIV-1 Gag. We calculate the depth of the attractive free energy potential between the MA/ CActd sites and find it to be about three times larger than the dimerization interaction between the CActd domains. Sequence analysis shows high conservation within the newly-found intra-Gag MA/CActd binding site, as well as its spatial proximity to other well known elements of Gag -such as CActd's SP1 helix region, its inositol hexaphosphate (IP6) binding site and major homology region (MHR), as well as the MA trimerization site. Our results point to a high, but yet undetermined, functional significance of the intra-Gag binding site. Recent biophysical experiments that address the binding specificity of Gag are interpreted in the context of the MA/CActd bound state, suggesting an important role in selective packaging of genomic RNA by Gag.

Endemic bacteriophages: a cautionary tale for evaluation of bacteriophage therapy and other interventions for infection control in animals.

BACKGROUND: One of the most effective targets for control of zoonotic foodborne pathogens in the farm to fork continuum is their elimination in food animals destined for market. Phage therapy for Escherichia coli O157:H7 in ruminants, the main animal reservoir of this pathogen, is a popular research topic. Since phages active against this pathogen may be endemic in host animals and their environment, they may emerge during trials of phage therapy or other interventions, rendering interpretation of trials problematic. METHODS: During separate phage therapy trials, sheep and cattle inoculated with 109 to 1010 CFU of E. coli O157:H7 soon began shedding phages dissimilar in plaque morphology to the administered therapeutic phages. None of the former was previously identified in the animals or in their environment. The dissimilar "rogue" phage was isolated and characterized by host range, ultrastructure, and genomic and proteomic analyses. RESULTS: The "rogue" phage (Phage vB_EcoS_Rogue1) is distinctly different from the administered therapeutic Myoviridae phages, being a member of the Siphoviridae (head: 53 nm; striated tail: 152x8 nm). It has a 45.8 kb genome which is most closely related to coliphage JK06, a member of the "T1-like viruses" isolated in Israel. Detailed bioinformatic analysis reveals that the tail of these phages is related to the tail genes of coliphage lambda. The presence of "rogue" phages resulting from natural enrichments can pose problems in the interpretation of phage therapeutic studies. Similarly, evaluation of any interventions for foodborne or other bacterial pathogens in animals may be compromised unless tests for such phages are included to identify their presence and potential impact.

Capture and alignment of phi29 viral particles in sub-40 nanometer porous alumina membranes.

Bacteriophage phi29 virus nanoparticles and its associated DNA packaging nanomotor can provide for novel possibilities towards the development of hybrid bio-nano structures. Towards the goal of interfacing the phi29 viruses and nanomotors with artificial micro and nanostructures, we fabricated nanoporous Anodic Aluminum Oxide (AAO) membranes with pore size of 70 nm and shrunk the pores to sub 40 nm diameter using atomic layer deposition (ALD) of Aluminum Oxide. We were able to capture and align particles in the anodized nanopores using two methods. Firstly, a functionalization and polishing process to chemically attach the particles in the inner surface of the pores was developed. Secondly, centrifugation of the particles was utilized to align them in the pores of the nanoporous membranes. In addition, when a mixture of empty capsids and packaged particles was centrifuged at specific speeds, it was found that the empty capsids deform and pass through 40 nm diameter pores whereas the particles packaged with DNA were mainly retained at the top surface of the nanoporous membranes. Fluorescence microscopy was used to verify the selective filtration of empty capsids through the nanoporous membranes.

Structure of native and expanded sobemoviruses by electron cryo-microscopy and image reconstruction.

Rice yellow mottle virus (RYMV) and southern bean mosaic virus, cowpea strain (SCPMV) are members of the Sobemovirus genus of RNA-containing viruses. We used electron cryo-microscopy (cryo-EM) and icosahedral image analysis to examine the native structures of these two viruses at 25 A resolution. Both viruses have a single tightly packed capsid layer with 180 subunits assembled on a T=3 icosahedral lattice. Distinctive crown-like pentamers emanate from the 12 5-fold axes of symmetry. The exterior face of SCPMV displays deep valleys along the 2-fold axes and protrusions at the quasi-3-fold axes. While having a similar topography, the surface of RYMV is comparatively smooth. Two concentric shells of density reside beneath the capsid layer of RYMV and SCPMV, which we interpret as ordered regions of genomic RNA. In the presence of divalent cations, SCPMV particles swell and fracture, whereas the expanded form of RYMV is stable. We previously proposed that the cell-to-cell movement of RYMV in xylem involves chelation of Ca(2+) from pit membranes of infected cells, thereby stabilizing the capsid shells and allowing a pathway for spread of RYMV through destabilized membranes. In the context of this model, we propose that the expanded form of RYMV is an intermediate in the in vivo assembly of virions.

Ultrastructural localization of nonstructural and coat proteins of 19 potyviruses using antisera to bacterially expressed proteins of plum pox potyvirus.

Antisera to the bacterially expressed nonstructural proteins (NSP) HC-Pro, CI, NIa, and NIb and the coat protein (CP) of plum pox potyvirus (PPV) were used for analysing the composition of virus-induced cytoplasmic and nuclear inclusions by electron microscopy. The antisera reacted with NSP and CP of PPV on immunogold-labelled ultrathin sections. Antiserum to CP reacted with virions of seven out of 18 other potyviruses. CP was distributed throughout the cytoplasm of infected cells. Antisera to PPV NSP specifically reacted with virus-specific cytoplasmic and/or nuclear inclusions induced by 17 different potyviruses. NSP were furthermore localized in confined cytoplasmic areas in between complex accumulations of virus-specific inclusions. Cylindrical inclusions induced by the potyviruses were proven to consist of CI protein. Most other cytoplasmic or nuclear inclusions were shown to be composed of two or more NSP. An unexpected composition of virus-induced inclusions was observed for the crystalline nuclear inclusions of tobacco etch virus. Here, in addition to the expected presence of NIa and NIb, HC-Pro could be demonstrated. Furthermore, amorphous cytoplasmic inclusions induced by papaya ringspot virus contained the expected HC-Pro but additionally NIa, NIb and CI. Beet mosaic virus-induced nuclear inclusions ('satellite bodies') contained in their electron-dense matrix NIa, NIb, Hc-Pro and CI and in their lacunae CP in bundles of virion-like filaments. The results indicate that all cytoplasmic or nuclear inclusions of potyviruses have to be regarded as deposition sites of excessively produced viral NSP.

Structure of Sesbania mosaic virus at 3 A resolution.

Sesbania mosaic virus (SMV) is an isometric, ss-RNA plant virus found infecting Sesbania grandiflora plants in fields near Tirupathi, South India. The virus particles, which sediment at 116 S at pH 5.5, swell upon treatment with EDTA at pH 7.5 resulting in the reduction of the sedimentation coefficient to 108 S. SMV coat protein amino acid sequence was determined and found to have approximately 60% amino acid sequence identity with that of southern bean mosaic virus (SBMV). The amino terminal 60 residue segment, which contains a number of positively charged residues, is less well conserved between SMV and SBMV when compared to the rest of the sequence. The 3D structure of SMV was determined at 3.0 A resolution by molecular replacement techniques using SBMV structure as the initial phasing model. The icosahedral asymmetric unit was found to contain four calcium ions occurring in inter subunit interfaces and three protein subunits, designated A, B and C. The conformation of the C subunit appears to be different from those of A and B in several segments of the polypeptide. These observations coupled with structural studies on SMV partially depleted of calcium suggest a plausible mechanism for the initiation of the disassembly of the virus capsid.