Structural changes associated with poliovirus inactivation in soil.

The loss of infectivity of poliovirus in moist and dried soils was a result of irreversible damage to the virus particles. The damage included (i) dissociation of viral genomes and capsids and (ii) degradation of viral ribonucleic acid (RNA) in the soil environment. Under drying conditions, capsid components could not be recovered from the soils. Further studies in sterile soils indicated that, under moist conditions, the viral RNA was probably damaged before dissociation from the capsid. However, in sterile, dried soil, RNA genomes were recovered largely intact from the soil. These results suggest that polioviruses are inactivated by different mechanisms in moist and drying soils.     

Structural changes associated with poliovirus inactivation in soil.

The loss of infectivity of poliovirus in moist and dried soils was a result of irreversible damage to the virus particles. The damage included (i) dissociation of viral genomes and capsids and (ii) degradation of viral ribonucleic acid (RNA) in the soil environment. Under drying conditions, capsid components could not be recovered from the soils. Further studies in sterile soils indicated that, under moist conditions, the viral RNA was probably damaged before dissociation from the capsid. However, in sterile, dried soil, RNA genomes were recovered largely intact from the soil. These results suggest that polioviruses are inactivated by different mechanisms in moist and drying soils.     

Infectious entry of West Nile virus occurs through a clathrin-mediated endocytic pathway

The pathway of West Nile flavivirus early internalization events was mapped in detail in this study. Overexpression of dominant-negative mutants of Eps15 strongly inhibits West Nile virus (WNV) internalization, and pharmacological drugs that blocks clathrin also caused a marked reduction in virus entry but not caveola-dependent endocytosis inhibitory agent, filipin. Using immunocryoelectron microscopy, WNV particles were seen within clathrin-coated pits after 2 min postinfection. Double-labeling immunofluorescence assays and immunoelectron microscopy performed with anti-WNV envelope or capsid proteins and cellular markers (EEA1 and LAMP1) revealed the trafficking pathway of internalized virus particles from early endosomes to lysosomes and finally the uncoating of the virus particles. Disruption of host cell cytoskeleton (actin filaments and microtubules) with cytochalasin D and nocodazole showed significant reduction in virus infectivity. Actin filaments are shown to be essential during the initial penetration of the virus across the plasma membrane, whereas microtubules are involved in the trafficking of internalized virus from early endosomes to lysosomes for uncoating. Cells treated with lysosomotropic agents were largely resistant to infection, indicating that a low-pH-dependent step is required for WNV infection. In situ hybridization of DNA probes specific for viral RNA demonstrated the trafficking of uncoated viral RNA genomes to the endoplasmic reticulum.     

Conformations, interactions, and thermostabilities of RNA and proteins in bean pod mottle virus: investigation of solution and crystal structures by laser Raman spectroscopy.

We report and interpret laser Raman spectra of the three virion components of bean pod mottle virus (BPMV). The top component of BPMV is an empty capsid; middle and bottom components package the RNA2 and RNA1 genome segments, respectively. All components were investigated as both single crystals and aqueous solutions, the latter over wide ranges of temperature and ionic strength. The isolated RNA2 molecule of BPMV middle component was also investigated in both H2O and D2O solutions. The results permit assessment of RNA and protein structures, their mutual interactions in the virions, and their conformational thermostabilities and comparison of these structural characteristics for solution and crystal states of the particles. The principal findings of this study are (i) The extent of ordered A-form backbone (74%) and of base pairing (38% AU + 22% GC) in unpackaged (aqueous) RNA2 are significantly altered by packaging. The A-form secondary structure of RNA2 is increased by 12 +/- 4%, and guanine base interactions are also substantially increased with packaging. (ii) The thermostability of Raman-monitored secondary structure of unpackaged RNA2 (Tm approximately 43 degrees C) is greatly increased in the packaged state (Tm approximately 53 degrees C). This increase corresponds to a stabilization of the A-form backbone geometry in 15 +/- 5% of genome nucleotides. (iii) Packaging of RNA2 in the middle component stabilizes subunit-subunit interactions of the capsid, as evidenced by a thermal denaturation temperature Td approximately 65 degrees C for the virion, compared with Td approximately 55 degrees C for the empty capsid. (iv) Raman marker-band shifts implicate the purine 7N sites of RNA2 and aromatic side chains of subunits as the principal targets for RNA-subunit interaction. (v) At the conditions of the present experiments (8 degrees C, pH approximately 7, moderate ionic strength), the subunit secondary structures observed for solutions of the top, middle, and bottom components are indistinguishable by Raman spectroscopy from secondary structures observed for corresponding crystalline samples. (vi) On the other hand, side chains of subunits in the top component (empty capsid) yield significantly different Raman intensities in crystalline and solution states. These differences are interpreted as the result of changes in a small number of side-chain environments between crystal and solution. (vii) Similarly, small differences exist between RNA Raman markers of crystalline and aqueous virions, which are attributed to altered environments of nucleotide residues and to a small increase in the amount of A-form backbone geometry upon going from the crystal to the solution.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of rifampicin on the infectivity of RNA bacteriophage f2.

RNA bacteriophage f2, treated in vitro with rifampicin, loses infectivity dramatically. Rifampicin interacts with phage RNA, binding to a few specific sites. Inhibition of phage RNA infectivity occurs at 10-100 times lower molar excess of rifampicin than inhibition of infectivity of intact phage particles. Thus the phage capsid acts as a barrier, diminishing interaction of the drug with phage RNA.     

Reconstitution of Flock House provirions: a model system for studying structure and assembly.

Assembly of Flock House virus in infected Drosophila cells proceeds through an intermediate, the provirion, which lacks infectivity until the coat precursor protein, alpha, undergoes a spontaneous "maturation" cleavage (A. Schneemann, W. Zhong, T. M. Gallagher, and R. R. Rueckert, J. Virol 6:6728, 1992). We describe here methods for purifying provirions in a state which permitted dissociation and reassembly. Dissociation, to monomeric alpha protein and free RNA, was accomplished by freezing at pH 9.0 in the presence of 0.5 M salt and 0.1 M urea. When dialyzed at low ionic strength and pH 6.5, the dissociation products reassembled spontaneously to form homogeneous provirions with a normal complement of RNA as judged by cosedimentation with authentic virions and by ability to undergo maturation cleavage with acquisition of substantial, though subnormal, infectivity. Reconstitution experiments, i.e., remixing components after separating RNA from capsid protein, generated abnormal particles, suggesting the presence in the unfractionated dissociation products of an unidentified "nucleating" component.     

Alteration of capsid proteins of coxsackievirus A13 by low ionic concentrations.

Several group A coxsackieviruses (A13, 15, 18, and 21), but not polioviruses or group B coxsackieviruses, are rapidly inactivated in low ionic strength solutions at neutral pH. The extent of inactivation is dependent upon temperature and molarity. Virions inactivated in this manner contain a normal complement of infectious RNA which remains in a state resistant to the action of ribonuclease. However, more than 95% of the virus particles are unable to attach to susceptible cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that coxsackievirus A13 virions contain five structural polypeptides (VP1, VP2a, VP2b, VP3, and VP4). Electrophoretic analysis indicates that inactivation of coxsackievirus A13 in low ionic strength solutions is due to the specific loss of the smallest polypeptide VP4 from the virus particle. These results suggest that adsorption of coxsackievirus A13 to receptors on susceptible cells is dependent upon the presence of the capsid protein VP4.     

Physical, Biochemical, and Immunological Properties of Coliphage MS-2 Particles

H (heavy) and L (light) MS-2 particles differ in density, absorption spectrum, and infectivity. Studies on their sedimentation, ribonucleic acid (RNA) content and infectivity, appearance under the electron microscope, ribonuclease sensitivity, and A-protein content failed to demonstrate any difference between the two particle types. Studies on the size, RNA content, and density of the capsid and two smaller coat protein components were also conducted. The antigenic relatedness of five different viral and subviral particles of MS-2 were studied by using immunodiffusion and neutralization. Capsids and the H and L viral particles were shown to be antigenically related, whereas the coat protein monomers and dimers were shown to be unrelated to the higher-molecular-weight particles.     

Bacteriophage inactivation at the air-water-solid interface in dynamic batch systems

Bacteriophages have been widely used as surrogates for human enteric viruses in many studies on virus transport and fate. In this investigation, the fates of three bacteriophages, MS2, R17, and phiX174, were studied in a series of dynamic batch experiments. Both MS2 and R17 readily underwent inactivation in batch experiments where solutions of each phage were percolated through tubes packed with varying ratios of glass and Teflon beads. MS2 and R17 inactivation was the result of exposure to destructive forces at the dynamic air-water-solid interface. phiX174, however, did not undergo inactivation in similar studies, suggesting that this phage does not accumulate at air-water interfaces or is not affected by interfacial forces in the same manner. Other batch experiments showed that MS2 and R17 were increasingly inactivated during mixing in polypropylene tubes as the ionic strength of the solution was raised (phiX174 was not affected). By the addition of Tween 80 to suspensions of MS2 and R17, phage inactivation was prevented. Our data suggest that viral inactivation in simple dynamic batch experiments is dependent upon (i) the presence of a dynamic air-water-solid interface (where the solid is a hydrophobic surface), (ii) the ionic strength of the solution, (iii) the concentration of surface active compounds in the solution, and (iv) the type of virus used.     

Rescue of maturation-defective flock house virus infectivity with noninfectious, mature, viruslike particles

The infectivity of flock house virus (FHV) requires autocatalytic maturation cleavage of the capsid protein at residue 363, liberating the C-terminal 44-residue γ peptides, which remain associated with the particle. In vitro studies previously demonstrated that the amphipathic, helical portion (amino acids 364 to 385) of γ is membrane active, suggesting a role for γ in RNA membrane translocation during infection. Here we show that the infectivity of a maturation-defective mutant of FHV can be restored by viruslike particles that lack the genome but undergo maturation cleavage. We propose that the colocalization of the two defective particle types in an entry compartment allows the restoration of infectivity by γ.     

Effect of macromolecular crowding agents on human immunodeficiency virus type 1 capsid protein assembly in vitro

Previous studies on the self-assembly of capsid protein CA of human immunodeficiency virus type 1 (HIV-1) in vitro have provided important insights on the structure and assembly of the mature HIV-1 capsid. However, CA polymerization in vitro was previously observed to occur only at very high ionic strength. Here, we have analyzed the effects on CA assembly in vitro of adding unrelated, inert macromolecules (crowding agents), aimed at mimicking the crowded (very high macromolecular effective concentration) environment within the HIV-1 virion. Crowding agents induced fast and efficient polymerization of CA even at low (close to physiological) ionic strength. The hollow cylinders thus assembled were indistinguishable in shape and dimensions from those formed in dilute protein solutions at high ionic strength. However, two important differences were noted: (i) disassembly by dilution of the capsid-like particles was undetectable at very high ionic strength, but occurred rapidly at low ionic strength in the presence of a crowding agent, and (ii) a variant CA from a presumed infectious HIV-1 with mutations at the CA dimerization interface was unable to assemble at any ionic strength in the absence of a crowding agent; in contrast, this mutation allowed efficient assembly, even at low ionic strength, when a crowding agent was used. The use of a low ionic strength and inert macromolecules to mimic the crowded environment inside the HIV-1 virion may lead to a better in vitro evaluation of the effects of conditions, mutations or/and other molecules, including potential antiviral compounds, on HIV-1 capsid assembly, stability and disassembly.     

Poliovirus Mutants at Histidine 195 of VP2 Do Not Cleave VP0 into VP2 and VP4

The final stage of poliovirus assembly is characterized by a cleavage of the capsid precursor protein VP0 into VP2 and VP4. This cleavage is thought to be autocatalytic and dependent on RNA encapsidation. Analysis of the poliovirus empty capsid structure has led to a mechanistic model for VP0 cleavage involving a conserved histidine residue that is present in the surrounding environment of the VP0 cleavage site. Histidine 195 of VP2 (2195H) is hypothesized to activate local water molecules, thus initiating a nucleophilic attack at the scissile bond. To test this hypothesis, 2195H mutants were constructed and their phenotypes were characterized. Consistent with the requirement of VP0 cleavage for poliovirus infectivity, all 2195H mutants were nonviable upon introduction of the mutant genomes into HeLa cells. Replacement of 2195H with threonine or arginine resulted in the assembly of a highly unstable 150S virus particle. Further analyses showed that these particles contain genomic RNA and uncleaved VP0, criteria associated with the provirion assembly intermediate. These data support the involvement of 2195H in mediating VP0 cleavage during the final stages of virus assembly.     

A circular dichroism study of the Turnip yellow mosaic virus-RNA

We examined the circular dichroism spectra of intact Turnip yellow mosaic virus, freezed/thawed virus, empty capsid particles, and phenol extracted RNA. The circular dichroism signal of the empty capsid was found to contribute for less than 1% to the circular dichroism of the virus. Differences in the circular dichroism spectra indicate that TYMV-RNA exhibits different conformations when it is in situ in the virus, when it has been ejected by freezing/thawing and when it has been phenol extracted. Increase of the ionic strength up to 0.1 M NaCl led to conformational change of the RNA either freezed/thawed ejected or phenol extracted but not in situ in the capsid. Addition of spermidine (3 mM) induced a conformational change only for the phenol extracted RNA. These results are discussed with respect to the origin of the various conformational states of viral RNA.     

Effect of ion binding on protein transport through ultrafiltration membranes

Electrostatic interactions can have a significant impact on protein transmission through semipermeable membranes. Experimental data for the transport of bovine serum albumin (BSA) through a polyethersulfone ultrafiltration membrane were obtained in different salt solutions over a range of pH and salt concentrations. Net BSA charge under the same conditions was evaluated from mobility data measured by capillary electrophoresis. The results show that specific ionic composition, in addition to solution pH and ionic strength, can strongly affect the rate of protein transport through semipermeable ultrafiltration membranes. The effects of different ions on BSA sieving are due primarily to differences in ion binding to the protein, which leads to significant differences in the net protein charge at a given pH and ionic strength. This effect could be described in terms of an effective protein radius, which accounts for the electrostatic exclusion of the charged protein from the membrane pores. These results provide important insights into the nature of the electrostatic interactions in membrane systems.     

Simple rules for efficient assembly predict the layout of a packaged viral RNA

Single-stranded RNA (ssRNA) viruses, which include major human pathogens, package their genomes as they assemble their capsids. We show here that the organization of the viral genomes within the capsids provides intriguing insights into the highly cooperative nature of the assembly process. A recent cryo-electron microscopy structure of bacteriophage MS2, determined with only 5-fold symmetry averaging, has revealed the asymmetric distribution of its encapsidated genome. Here we show that this RNA distribution is consistent with an assembly mechanism that follows two simple rules derived from experiment: (1) the binding of the MS2 maturation protein to the RNA constrains its conformation into a loop, and (2) the capsid must be built in an energetically favorable way. These results provide a new level of insight into the factors that drive efficient assembly of ssRNA viruses in vivo.     

Procedure for the Preparation of Milligram Quantities of Adenovirus Messenger Ribonucleic Acid

Late after adenovirus 2 infection (18 hr), nearly all newly synthesized polysomal messenger ribonucleic acid (mRNA) is viral specified. Large amounts of adenovirus mRNA have been purified by utilizing membrane filtration at high ionic strength. With this procedure, molecules that contain polyadenylic acid [poly (A)] tracts are bound selectively, and ribosomal RNA can be separated from the viral mRNA which contains poly(A). Polysomal RNA synthesized 18 hr after infection was labeled in the presence of 0.02 mug of actinomycin D per ml and extracted at pH 9.0. This RNA annealed 40% to 3 mug of adenovirus 2 deoxyribonucleic acid; the RNA selected by membrane filtration bound 80% under the same conditions. The RNA eluted from membrane filters was 80 to 90% greater than 18S and contained species migrating as 31, 27, and 24S. Binding of polysomal RNA to individual membrane filters was linear, using as much as 300 mug of RNA per membrane. A 1.1-mg amount of viral RNA was prepared from 17.7 mg of polysomal RNA that had been purified by extraction at pH 9.0.     

Use of sedimentation under conditions of acidic denaturation for the determination of molecular weights of RNAs]

The conditions for acidic denaturation of double stranded RNA were found. Under these conditions a limited degradation of high molecular weight viral RNA took place. This degradation was determined by the degree of fragmentation and loss of infectivity at acidic conditions. It was found that acidic denaturation of RNA in the solutions of low ionic strength was accompanied by a considerable increase of sedimentation coefficient. Under these conditions the coefficients of sedimentation and molecular weights of RNAs studied are connected by the following function S20=2.84-10(-2) Mr0.689. The conclusion has been drawn that the sedimentation under the conditions for acidic denaturation could be used both for molecular weight determination and the practical preparation of unaggregated strands of RNA.