Effects of Virion and Salt Concentrations on the Raman Signatures of Filamentous Phages fd, Pf1, Pf3, and PH75

Abstract

Filamentous phages consist of a single-stranded DNA genome encapsidated by several thousand copies of a small alpha-helical coat protein subunit plus several copies of four minor proteins at the filament ends. The filamentous phages are important as cloning vectors, vehicles for peptide display, and substrates for macromolecular alignment. Effective use of a filamentous phage in such applications requires an understanding of experimental factors that may influence the propensity of viral filaments to laterally aggregate in solution. Because the Raman spectrum of a filamentous phage is strongly dependent on the relative orientation of the virion with respect to the polarization direction of the electromagnetic radiation employed to excite the spectrum, we have applied Raman spectroscopy to investigate lateral aggregation of phages fd, Pf1, Pf3, and PH75 in solution. The results show that lateral aggregation of the virions and anisotropic orientation of the aggregates are both disfavored by high concentrations of salt (>200 mM NaCl) in solutions containing a relatively low virion concentration (<10 mg/mL). Conversely, the formation of lateral aggregates and their anisotropic orientation are strongly favored by a low salt concentration (<0.1 mM NaCl), irrespective of the virion concentration over a wide range. The use of Raman polarization effects to distinguish isotropic and anisotropic solutions of filamentous phages is consistent with previously reported Raman analyses of virion structures in both solutions and fibers. The Raman data are supported by electron micrographs of negatively stained specimens of phage fd, which permit an independent assessment of salt effects on lateral aggregation. The present results also identify new Raman bands that serve as potential markers of subunit side-chain orientations in filamentous virus assemblies.

Raman, polarized Raman and ultraviolet resonance Raman spectroscopy of nucleic acids and their complexes

“…Excitation at 257 nm results in a UVRR spectrum dominated by viral DNA, while excitation at 229 nm yields a UVRR profile primarily of tryptophan and tyrosine residues of the capsid subunit, as labeled. 74,159,198,199 52 Earlier studies report scattering cross-sections for several additional UV excitation wavelengths. 68,69 Spectral data collection and processing Raman spectra of nucleic acids exhibiting acceptable signalto-noise ratios (¾10 : 1 or greater for the most intense bands in the spectrum) are typically generated by averaging independently collected sets of data on the same or identical samples.…”

Glycerol facilitates the disaggregation of recombinant adeno-associated virus serotype 2 on mica surface

“…Thus, how to prevent virus aggregation becomes an important scientific and technical issue. In pioneering work, the mechanisms and the kinetics of salts on viral disaggregation in suspension have been extensively investigated [5,6]. In some cases additives present in particle suspension may cause various effects on dispersing of the particles.…”

The Structure of a Filamentous Bacteriophage

“…12 Data were obtained from native specimens prepared under conditions that minimize aggregation. 13 Using 84,310 filament segments (each of length 240 Å) extracted from filaments in ice, we failed to achieve convergence to a common structure from different starting symmetries. This suggested structural polymorphism.…”