Assessment of the Applicability of Capsid-Integrity Assays for Detecting Infectious Norovirus Inactivated by Heat or UV Irradiation.

Human noroviruses are the leading cause of viral gastroenteritis. In the absence of a practical culture technique for routine analysis of infectious noroviruses, several methods have been developed to discriminate between infectious and non-infectious viruses by removing non-viable viruses prior to analysis by RT-qPCR. In this study, two such methods (RNase and porcine gastric mucin) which were designed to remove viruses with compromised capsids (and therefore assumed to be non-viable), were assessed for their ability to quantify viable F-specific RNA bacteriophage (FRNAP) and human norovirus following inactivation by UV-C or heat. It was found that while both methods could remove a proportion of non-viable viruses, a large proportion of non-viable virus remained to be detected by RT-qPCR, leading to overestimations of the viable population. A model was then developed to determine the proportion of RT-qPCR detectable RNA from non-viable viruses that must be removed by such methods to reduce overestimation to acceptable levels. In most cases, nearly all non-viable virus must be removed to reduce the log overestimation of viability to within levels that might be considered acceptable (e.g. below 0.5 log10). This model could be applied when developing alternative pre-treatment methods to determine how well they should perform to be comparable to established infectivity assays.

Production, purification, and capsid stability of rhinovirus C types.

The rhinovirus C (RV-C) were discovered in 2006 and these agents are an important cause of respiratory morbidity. Little is known about their biology. RV-C15 (C15) can be produced by transfection of recombinant viral RNA into cells and subsequent purification over a 30% sucrose cushion, even though yields and infectivity of other RV-C genotypes with this protocol are low. The goal of this study was to determine whether poor RV-C yields were due to capsid instability, and moreover, to develop a robust protocol suitable for the purification of many RV-C types. Capsid stability assays indicated that virions of RV-C41 (refractory to purification) have similar tolerance for osmotic and temperature stress as RV-A16 (purified readily), although C41 is more sensitive to low pH. Modification to the purification protocol by removing detergent increased the yield of RV-C. Addition of nonfat dry milk to the sucrose cushion increased the virus yield but sacrificed purity of the viral suspension. Analysis of virus distribution following centrifugation indicated that the majority of detectable viral RNA (vRNA) was found in pellets refractory to resuspension. Reduction of the centrifugal force with commiserate increase in spin-time improved the recovery of RV-C for both C41 and C2. Transfection of primary lung fibroblasts (WisL cells) followed by the modified purification protocol further improved yields of infectious C41 and C2. Described herein is a higher yield purification protocol suitable for RV-C types refractory to the standard purification procedure. The findings suggest that aggregation-adhesion problems rather than capsid instability influence RV-C yield during purification.

Capsid-Damaging Effects of UV Irradiation as Measured by Quantitative PCR Coupled with Ethidium Monoazide Treatment.

The damage to a viral capsid after low-pressure (LP) and medium-pressure (MP) UV irradiation was assessed, using the quantitative or quantitative reverse transcription PCR coupled with ethidium monoazide treatment (EMA-PCR). After UV irradiation, adenovirus 5 (Ad5) and poliovirus 1 (PV1) were subjected to a plaque assay, PCR, and EMA-PCR to investigate the effect of UV irradiation on viral infectivity, genome damage, and capsid damage, respectively. The effectiveness of UV wavelengths in a viral genome and capsid damage of both PV1 and Ad5 was also further investigated using a band-pass filter. It was found that an MPUV lamp was more effective than an LPUV lamp in inactivating Ad5, whereas there was no difference in the case of PV1. The results of viral reduction determined by PCR and EMA-PCR indicated that MP UV irradiation damaged Ad5 capsid. The damage to PV1 and Ad5 capsid was also not observed after LP UV irradiation. The investigation of effects of UV wavelengths suggested that UV wavelengths at 230-245 nm have greater effects on adenovirus capsid in addition to viral genome than UV wavelengths beyond 245 nm.

Biophysical and ultrastructural characterization of adeno-associated virus capsid uncoating and genome release.

We describe biophysical and ultrastructural differences in genome release from adeno-associated virus (AAV) capsids packaging wild-type DNA, recombinant single-stranded DNA (ssDNA), or dimeric, self-complementary DNA (scDNA) genomes. Atomic force microscopy and electron microscopy (EM) revealed that AAV particles release packaged genomes and undergo marked changes in capsid morphology upon heating in physiological buffer (pH 7.2). When different AAV capsids packaging ss/scDNA varying in length from 72 to 123% of wild-type DNA (3.4 to 5.8 kb) were incrementally heated, the proportion of uncoated AAV capsids decreased with genome length as observed by EM. Genome release was further characterized by a fluorimetric assay, which demonstrated that acidic pH and high osmotic pressure suppress genome release from AAV particles. In addition, fluorimetric analysis corroborated an inverse correlation between packaged genome length and the temperature needed to induce uncoating. Surprisingly, scAAV vectors required significantly higher temperatures to uncoat than their ssDNA-packaging counterparts. However, externalization of VP1 N termini appears to be unaffected by packaged genome length or self-complementarity. Further analysis by tungsten-shadowing EM revealed striking differences in the morphologies of ssDNA and scDNA genomes upon release from intact capsids. Computational modeling and molecular dynamics simulations suggest that the unusual thermal stability of scAAV vectors might arise from partial base pairing and optimal organization of packaged scDNA. Our work further defines the biophysical mechanisms underlying adeno-associated virus uncoating and genome release.

Structure and photoelectrochemistry of a virus capsid-TiO2 nanocomposite.

The use of protein templates to direct the formation of inorganic nanostructures offers a novel bio-inspired route to nanomaterial synthesis that avoids the use of harsh reaction conditions and offers unique functionalities including biocompatibility and hierarchical assembly. Pair distribution function (PDF) analysis from total X-ray scattering has been used to determine the structure of TiO2 nanoparticles grown within an icosahedral virus capsid. The protein-TiO2 composites are similar to nanocrystalline anatase and show photocatalytic activity. PDF analysis is ideally suited to the study of protein-inorganic nanocomposites, and may be able to provide information about the hard/soft interface.

Penton release from P22 heat-expanded capsids suggests importance of stabilizing penton-hexon interactions during capsid maturation.

Bacteriophage assembly frequently begins with the formation of a precursor capsid that serves as a DNA packaging machine. The DNA packaging is accompanied by a morphogenesis of the small round precursor capsid into a large polyhedral DNA-containing mature phage. In vitro, this transformation can be induced by heat or chemical treatment of P22 procapsids. In this work, we examine bacteriophage P22 morphogenesis by comparing three-dimensional structures of capsids expanded both in vitro by heat treatment and in vivo by DNA packaging. The heat-expanded capsid reveals a structure that is virtually the same as the in vivo expanded capsid except that the pentons, normally present at the icosahedral fivefold positions, have been released. The similarities of these two capsid structures suggest that the mechanism of heat expansion is similar to in vivo expansion. The loss of the pentons further suggests the necessity of specific penton-hexon interactions during expansion. We propose a model whereby the penton-hexon interactions are stabilized through interactions of DNA, coat protein, and other minor proteins. When considered in the context of other studies using chemical or heat treatment of capsids, our study indicates that penton release may be a common trend among double-stranded DNA containing viruses.

Mechanism of the photocatalytic inactivation of Lactobacillus casei phage PL-1 by titania thin film.

The mechanism of the inactivation of Lactobacillus casei phage PL-1 suspended in a phosphate buffer by black-light (BL) -catalytic titanium dioxide (TiO2) thin film was studied. Generation of both superoxide anions (O2-) and hydroxyl radicals (*OH) was confirmed in the aqueous medium in which TiO2 film was settled with BL irradiation under gentle shaking. With BL-irradiation alone without TiO2 film, only O2- was generated to some extent. The genome DNA inside the phage particles was found to be fragmented by the treatment of PL-1 phages with BL-catalytic TiO2 film. The phage inactivation by BL-catalytic TiO2 film was inhibited by the addition of albumin in a concentration-dependent manner. BL-catalytic TiO2 film was considered to cause primarily the damage to the capsid protein through the generation of active oxygen species such as *OH, followed by damage to the genome DNA inside the phage particles.

Preparation of oligoribonucleotides containing 4-thiouridine using Fpmp chemistry. Photo-crosslinking to RNA binding proteins using 350 nm irradiation.

The preparation of a 4-thiouridine phosphoramidite suitable for RNA synthesis and its subsequent incorporation into oligoribonucleotides is described. The thiol group is protected with a 2-cyanoethyl group and the 2'-OH with a 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl function. Thiouridine-containing oligoribonucleotides were used as 350 nm UV crosslinking probes for the photoaffinity labelling of RNA binding proteins. Specific crosslinking was demonstrated between the Rev protein of HIV-1 (as a glutathione S-transferase fusion protein) and its RNA target, the Rev-responsive element. It was not possible to generate crosslinks between the RNA bacteriophage MS2 coat protein and the initiator stem-loop of the replicase gene, to which it binds. These results are consistent with the structural data available on both systems.

A blocking ELISA for detection of antibody to a subgroup-reactive epitope of African horsesickness viral protein 7 (VP7) using a novel gamma-irradiated antigen.

A novel gamma irradiated inactivated cell culture derived African horsesickness viral (AHSV) antigen was used in a blocking ELISA (B-ELISA) for detecting antibody to a subgroup-reactive epitope of AHSV. A monoclonal antibody (MAB), class IgM, against an epitope on African horsesickness (AHS) viral protein 7 (VP7) was developed in BALBc mice and used in the B-ELISA. The MAB, designated F9H, was blocked by 69 serums from equidae with antibody to AHS, but its binding activity was not appreciably affected by 301 serums that did not contain antibodies to AHS virus. An ELISA protocol using a blocking format is described.

Analysis of viral DNA, protein and envelope damage after methylene blue, phthalocyanine derivative or merocyanine 540 photosensitization.

Although numerous photosensitizers have been used experimentally to decontaminate viruses in cellular blood components, little is known about their mechanisms of photoinactivation. Using M13 bacteriophage and vesicular stomatitis virus (VSV) as model viruses, we have investigated alteration of the viral genome, protein and envelope after phototreatment. Methylene blue (MB) and aluminum phthalocyanine tetrasulfonate (AlPcS4) phototreatment inactivated bacteriophage M13 and decreased the fraction of single-stranded circular genomic DNA (sc-DNA) by converting it to linear form. This conversion was enhanced by treating the extracted DNA with piperidine at 55 degrees C. Piperidine-labile breaks were well correlated to phage survival (5.1% sc-DNA at 1.7% phage survival for MB) under conditions where only minor differences were seen in the relative abundance of M13 coat protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Neither aluminum phthalocyanine (AlPc) nor merocyanine 540 (MC540) inactivated M13 nor were there significant changes observed in DNA and coat protein. Methylene blue, AlPcS4 and AlPc inactivated VSV and inhibited fusion of the virus envelope to Vero cells at pH 5.7 (i.e. with plasma membrane). However, the degree of this inhibition was small compared to the extent of virus inactivation (43% inhibition vs. 4.7 log10 or 99.998% inactivation, for MB). In contrast, an antibody to VSV G-spike protein inhibited fusion at pH 5.7 by 52% with a concomitant decline in VSV infectivity of 0.15 log10 (30%). Few changes were observed in the relative abundance of G protein for MB and AlPcS4 phototreated samples and no additional protein bands were observed on SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of radiation damage on the structure of frozen hydrated HSV-1 capsids.

Radiation damage imposes stringent limits on the information content of electron micrographs of biological specimens. In this study, we have investigated its effects on frozen, hydrated specimens and three-dimensional reconstructions calculated from cryomicrographs using capsids of herpes simplex virus as a model system. Multiple-exposure series of micrographs of both B-capsids (which contain no DNA) and C-capsids (which are fully packaged) were recorded and reconstructions were calculated from the first exposures, corresponding to a cumulative electron dose of 6-7 e-/A2, and from later exposures (25-40 e-/A2). Experimental procedures were standardized to ensure that perceived changes in the micrographs and reconstructions would be attributable to radiation damage alone. The effects of the higher doses in both the micrographs and the reconstructions were expressed as a progressive blurring of the finer details, corresponding to a delocalization of structure in the ice-embedded specimens. The resolutions of the reconstructions were quantified according to a form of the Fourier ring correlation coefficient criterion, according to which the first-exposure reconstructions had resolutions of 30-36 A. The fifth-exposure B-capsid reconstruction had comparable nominal resolution, although it exhibited progressively lower correlations at higher spatial frequencies. Qualitatively similar changes in the series of C-capsid reconstructions were observed although they were more pronounced, presumably because these micrographs had lower contrast and signal-to-noise ratios. We infer that the observed changes in the images and reconstructions and the concomitant loss in contrast in the immediate vicinity of the capsid surface may reflect radiation-induced perturbation of molecular structure and/or the release of peptide fragments. Nevertheless, the observed changes are relatively subtle, at least at the operational resolution of this study; overall, our results support earlier indications (M. F. Schmid et al. J. Struct. Biol. 108, 62-68, 1992) that prospects are quite good for tilt-series reconstructions from cryoelectron micrographs, including six to eight views of the same specimen.

Mutations that eliminate the requirement for the vertex protein in bacteriophage T4 capsid assembly.

The capsid of bacteriophage T4 is composed of two essential structural proteins, gp23, the major constituent of the capsid, and gp24, a less prevalent protein that is located in the pentameric vertices of the capsid. gp24 is required both to stabilize the capsid and to allow it to be further matured. This requirement can be eliminated by bypass-24 (byp24) mutations within g23. We have isolated, cloned and sequenced several new byp24 mutations. These mutations are cold-sensitive in the absence of gp24, and are located in regions of g23 not known to contain any other mutations affecting capsid assembly. The cold-sensitivity of the byp24 mutations can be reduced by further mutations within g23 (trb mutations). Cloning and sequencing of these trb mutations has revealed that they lie in regions of g23 that contain clusters of mutations that cause the production of high levels of petite and giant phage (ptg mutations). Despite the proximity of the trb mutations to the ptg mutations, none of the ptg mutations has a Trb phenotype. The mutation ptE920g, which is also located near one of the ptg clusters, and which produces only petite and wild-type phage, has been shown to confer a Trb but not a Byp24 phenotype. The relevance of these observations to our understanding of capsid assembly is discussed.

Conformation of DNA packaged in bacteriophage T7. Analysis by use of ultraviolet light-induced DNA-capsid cross-linking.

The conformation of the linear, double-stranded, 39,936 kilobase-pair DNA packaged in the protein capsid of bacteriophage T7 is investigated here by use of short wavelength ultraviolet light-induced DNA-capsid cross-linking. To detect both DNA-capsid and DNA-DNA cross-links, DNA is expelled from the T7 capsid and the products of expulsion are analyzed by use of Nycodenz buoyant density centrifugation, followed by either pulsed field gel electrophoresis or invariant field gel electrophoresis. Short wavelength ultraviolet light is found to progressively induce both DNA-DNA and DNA-protein cross-links in intact bacteriophage T7, but not in T7 from which DNA had been expelled before exposure to ultraviolet light. Protein-protein cross-links are not induced. When DNA expelled from previously cross-linked T7 is cleaved with restriction endonuclease (1 to 3 sites cleaved), analysis of the resulting fragments reveals no regions on T7 DNA that are excluded from cross-linking to the capsid. However, the efficiency of cross-linking decreases as the distance from the left end (last end packaged) of the packaged DNA increases. Electron microscopy of negatively stained capsid-DNA complexes reveals no DNA-retaining structure other than the outer shell of the capsid. Together with previously reported data that indicate lack of protein-based specificity for ultraviolet light-induced cross-linking, these observations are interpreted by the assumptions that, within the limits of resolution of these experiments: (1) no region of packaged T7 DNA is excluded from contact with the outer shell of the T7 capsid; (2) the probability of contacting the outer shell decreases as the distance from the left end of packaged T7 DNA increases. Thus, T7 DNA packaging concentrates the last end packaged near the inner surface of the outer shell of the T7 capsid.

[Identification of a peptide of the membrane protein of tobacco mosaic virus, cross-linked with intraviral RNA under the effect of UV-radiation]. / Identifikatsiia peptida belka obolochki virusa tabanoi mozaiki, sshivaiushchegosiia s vnutrivirusnoi RNK pod deistviem UF- izlucheniia.

As reported previously, UV-irradiation induces crosslinking between tobacco mosaic virus (TMV) coat protein molecules and intraviral RNA nucleotides. We have irradiated [3H]-uridine labeled TMV and isolated TMV coat protein subunits with the attached nucleotide label. These TMV protein subunits were hydrolyzed with trypsin. The tryptic peptides were separated by high-performance liquid chromatography and [3H]-labeled peptides were identified. The UV-irradiation of TMV was found to result in crosslinking to intraviral RNA of the T8 tryptic peptide (residues 93-112) of TMV coat protein.

Alteration of vesicular stomatitis virus L and NS proteins by uv irradiation: implications for the mechanism of host cell shut-off.

When purified, [35S]methionine-labeled vesicular stomatitis virus (VSV) was exposed to ultraviolet light, an irradiation-induced change in the viral proteins was detected by SDS-polyacrylamide gel electrophoresis and immunoblotting. With dose of uv irradiation in the same range as that required to inactivate VSV leader RNA, a loss occurred in the bands corresponding to the L and NS proteins concomitant with the appearance of several new bands of radioactivity throughout the gel. This alteration of viral proteins correlated with the loss of ability of the virus to inhibit host macromolecular synthesis. In light of these results, the role that has been ascribed to the VSV leader RNA in VSV-mediated host shut-off needs to be reevaluated.

Interferon induction by adenovirus type 12: stimulatory function of early region 1A.

Adenoviruses are generally weak interferon inducers, triggering chicken embryo fibroblast cells by a UV-resistant viral component, probably the capsid or capsid elements, to produce 50 to 100 IU of interferon per ml. Adenovirus types 12, 18, and 31, however, can induce by a UV-sensitive mechanism 10 to 20 times more interferon than other types do. By using mutant and recombinant adenoviruses, we demonstrated that early region 1A was responsible for the enhanced interferon production of chicken cells infected with adenovirus type 12.

UV irradiation impairs in vivo encapsidation of bacteriophage T4 DNA.

T4 DNA structural requirements for encapsidation in vivo were investigated, using thin-section electron microscopy to quantitate the kinetics and yields of head intermediates after synchronous DNA packaging into accumulated processed proheads. UV irradiation (254 nm) of T4-infected bacteria just before initiation of encapsidation resulted in a reduction in the rate of DNA packaged measured by electron microscopy and in the yield of viable phage progeny. In UV-irradiated infections with excision-deficient mutants (denV-), the extent of packaging decline was proportional to the UV dose and phage yields were lower than expected based on the packaging levels observed by microscopy. Rescue analysis of progeny from such infections revealed elevated levels of nonviable virions. Pyrimidine dimers were encapsidated in denV- infections, but in excision-competent infections (denV+) dimers were not packaged. A UV-independent, 15 to 20% packaging arrest was also observed when denV endonuclease was inactive during encapsidation, indicating a denV requirement to achieve normal T4 packaging levels. Pyrimidine dimers apparently represent or induce transient blockage of DNA encapsidation or both, causing a decline in the rate. This is in contrast to other DNA structural blocks to packaging induced by mutations in T4 genes 30 and 49, which appear to arrest the process.

Contributions of various types of damage to inactivation of T4 bacteriophage by protons.

Mean doses for damage induced by 3.7-MeV protons in T4 phage were measured for the following effects: inactivation, killing, adsorption, DNA injection, capsid rupture with DNA release, and single- and double-strand DNA breaks. These effects have been related to phage survival in the same experiment because of the variability inherent in such measurements. The experiments were carried out in nutrient broth, phosphate buffer, and phosphate buffer plus histidine as suspension media. The following conclusions can be drawn: (i) DNA double-strand breakage is the dominant cause of inactivation in nutrient broth; (ii) scavengers protect the DNA inside the capsid to only a small degree; (iii) indirect actions affect functions associated with proteins; (iv) DNA release, as measured by capsid rupture, accounts for only a small percentage of the loss of viability; (v) essentially all DNA from adsorbed phage is injected even though a large proportion of the DNA contains double-strand breaks.

Study of negatively stained images of Sendai virus nucleocapsids using minimum-dose system.

Using minimum-dose system and optical diffraction, effects of electron irradiation on negatively stained images of trypsin-straightened nucleocapsids of Sendai virus were semiquantitatively compared for uranyl-acetate (UA) and phosphotungstic acid (PTA). The results confirmed the superiority of UA in display of fine structures and showed that both UA- and PTA-stained images tended to turn from a one-sided to a two-sided image during irradiation, the general contrast of the picture increased in the UA-stained images but not in the PTA-stained ones, and furthermore the electron doses for the richest information were 18 000 to 30 000 e-/nm2 for UA, but 1000 e-/nm2 for PTA, under the condition used. The optical diffraction patterns of the UA-stained nucleocapsids, its analysis by the superposition method, and rotational harmonics of end-on views of nucleocapsids, together indicated that the most probable arrangement of subunits was 13 per turn of a helix with 5-nm periodicity. This helix also had an arrangement of subunits parallel to the axis. The occurrence of 2.5 nm periodicity was probably produced by an arrangement of a UA-penetrable concave substructure of the subunit.

Protein-RNA interaction in encephalomyocarditis virus as revealed by UV light-induced covalent linkages.

UV irradiation of encephalomyocarditis virus led to an increase in the buoyant density of the virus in CsCl gradients from 1.34 to 1.46 g/cm3. Heat treatment of the irradiated virus (20 min at 54 degrees C) reduced the density to 1.40 g/cm3 and led to the loss of approximately 55% of the labeled RNA from the virions. The non-irradiated virions were converted by such heating into empty capsids. Irradiation also resulted in an increase in the accessibility of RNA inside the virions to the action of pancreatic RNase. An increase in the UV dose did not enlarge the fraction of RNA molecules covalently linked to protein; this was revealed by the lack of any secondary increase in the apparent RNase resistance of the labeled RNA in the irradiated virions. Destruction of the irradiated virus with sodium dodecyl sulfate and 2-mercaptoethanol allowed the isolation of a 40S structure containing viral RNA and RNA-linked proteins. The latter comprised no more than 2.5% of the whole protein content of the virion. Polyacrylamide gel electrophoretic analysis of the RNase-treated 40S structure revealed at least three viral structural proteins in the same ratio as was present in the intact virions.