The organization of potato virus X coat proteins in virus particles studied by tritium planigraphy and model building

Vibrational circular dichroism (VCD) spectra in the amide I and II regions have been measured for viruses for the first time. VCD spectra were recorded for films prepared from aqueous buffer solutions and also for solutions using D 2 O buffers at pH 8. Investigations of four filamentous plant viruses, Tobacco mosaic virus (TMV), Papaya mosaic virus, Narcissus mosaic virus (NMV) and Potato virus X (PVX), as well as a deletion mutant of PVX, are described in this paper. The film VCD spectra of the viruses clearly revealed helical structures in the virus coat proteins; the nucleic acid bases present in the single-stranded RNA could also be characterized. In contrast, the solution VCD spectra showed the characteristic VCD bands for a-helical structures in the coat proteins but not for RNA. Both sets of results clearly indicated that the coat protein conformations are dominated by helical structures, in agreement with earlier reports. VCD results also indicated that the coat protein structures in PVX and NMV are similar to each other and somewhat different from that of TMV. The present study demonstrates the feasibility of measuring VCD spectra for viruses and extracting structural information from these spectra. INTRODUCTIONA variety of different spectroscopic and diffraction techniques have been used for structural analyses of viruses. These techniques include electronic circular dichroism (ECD) in the UV-visible region, vibrational Raman optical activity (ROA), crystallography for spherical viruses and fibre diffraction for filamentous viruses. While crystallography and fibre diffraction have the potential to provide complete descriptions of virus structures, they depend on the availability of virions at high purity and in relatively large quantities, and the formation of well-ordered crystals or fibres. They are therefore often not practical sources of structural information. The spectra structure correlation in ECD spectroscopy depends on observing characteristic ECD bands associated with a particular structure (Homer & Goodman, 1975;Erickson et al., 1981). For proteins, such correlation can sometimes be misleading because of the overlap of ECD bands originating from the peptide backbone with those from aromatic groups. Specifically for viruses, differential light scattering, which is proportional to the fourth power of incident wavelength, can interfere with ECD bands. Furthermore, ECD spectroscopy is known to yield artefact signals when film samples are studied. ROA spectroscopy (Barron, 2004; Blanch et al., 2002) is ideal for aqueous solution samples, but fluorescence interference and the requirement for higher concentrations pose limitations in some cases. Also it has not yet been possible to undertake ROA spectroscopy for film samples. The abovementioned limitations can be avoided with vibrational circular dichroism (VCD) spectroscopy.VCD is a measure of the difference in absorption for left and right circularly polarized light in the infrared (IR) region (Keiderling, 1981;Nafie, 1984;Polavarapu, 1998;Barr...

Section: Resultsmentioning

confidence: 99%

Structures of plant viruses from vibrational circular dichroism

Shanmugam

Polavarapu

Kendall

et al. 2005

“…It is unclear whether the mutated CP has a defect in CP-RNA interactions or in protein-protein interactions during the assembly process. According to the model proposed for the PVX CP by Baratova et al (1992) the deleted amino acid is found within an area of the CP that does not contain a-helical or fl-structural regions involved in the folding of the CP.…”

Section: Discussionmentioning

confidence: 99%

Infectious RNA transcripts derived from cloned cDNA of papaya mosaic virus: effect of mutations to the capsid and polymerase proteins

Sit

AbouHaidar

1993

Genomic length cDNAs of papaya mosaic virus (PMV)RNA were generated utilizing reverse transcriptase (RNase H-) for first strand synthesis, Sequenase for second strand synthesis and primers specific for the 5' and 3' termini of the viral genome. These cDNAs were cloned into plasmid pUC18 and infectious RNA transcripts were synthesized in vitro from a bacteriophage T7 RNA polymerase promoter incorporated into the 5' specific primer. The infectivity of transcripts was 16 % that of native PMV RNA. Increasing the poly(A) tail length from A24 to A71 produced a 43 % increase in infectivity. Transcripts synthesized with or without an m7GpppG cap structure were biologically active although uncapped transcripts were much less infectious. The addition of up to 2434 non-viral nucleotides at the 3' end of transcripts decreased but did not abolish infectivity. Insertions of two amino acid residues within the polymerase coding region inactivated viral transcripts. A single amino acid deletion within the capsid protein (CP) produced local lesions of a reduced size as compared to native PMV RNA. Viral particles could not be observed in crude extracts from lesions produced by this deletion mutant suggesting that it exists as a naked RNA species within the host. Mutations to the CP suggest that it is required not only for viral assembly but also for some other unidentified function(s) during the replication cycle.

“…Another likely result of the TGB1-induced virion structural transition might be virion conversion to a movement-competent form. One can speculate that after TGB1-induced structural transition this protein region might also appear to be located on the outer surface of transport-competent TGB1-bound virions similar to the localization of C-terminal PVX CP region to the outer virion surface upon protease treatment (Baratova et al, 1992), and to directly participate in interactions required for viral movement. It should be kept in mind that potexvirus cell-to-cell transport is coupled with the replication of viral RNA (Tilsner et al, 2013).…”

Section: Role Of Virions In Cell-to-cell Transport Of Filamentous Virmentioning

confidence: 99%

“…The mechanisms of translational activation induced by phosphorylation and TGB1 binding are seemingly different; however, the N-terminal domain of PVX CP can influence the conformational state of the C-terminal region of the CP in the virion. Indeed, in particles with the 19-21 N-terminal CP residues removed by proteolysis, the C-terminal region, which normally faces the inner virion channel, is now located at the outer virion surface (Baratova et al, 1992). In the context of this review, the ability of the N-terminal CP region to determine structural characteristics of virion CP molecules and mediate, upon phosphorylation or deletion, their conformational switch (changes in inter-subunit contacts and/or conformational alteration of a peptide backbone) is most significant, demonstrating that external stimuli can influence the structure of filamentous virions and thereby emphasizing their structural lability.…”

Section: Translational Activation Of Helical Virionsmentioning

confidence: 99%

“…In view of these technical issues, alternative indirect methods were used. Among those, 'tritium planigraphy' was employed to identify the CP regions exposed on the surface of PVX virions (Baratova et al, 1992(Baratova et al, , 2004. These data, combined with the data obtained with other indirect approaches, were used to develop a model for the PVX CP structural fold (Nemykh et al, 2008).…”

Section: Progress In Structural Studies Of Helical Virusesmentioning

confidence: 99%

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Helical capsids of plant viruses: architecture with structural lability

Solovyev

Makarov

2016

Capsids of numerous filamentous and rod-shaped plant viruses possess helical symmetry. In positive-stranded RNA viruses, helical capsids are typically composed of many identical subunits of the viral capsid protein (CP), encapsidating a molecule of viral genomic RNA. Current progress in structural studies of helical plant viruses has revealed differences between filamentous and rod-shaped viruses, both in structural folds of their CPs and in the interactions of CP molecules in their capsids. Many filamentous and rod-shaped viruses have functionally similar lateral inter-subunit contacts on the outer virion surface. Additionally, the extreme Nterminal CP region in filamentous viruses is intrinsically disordered. Taken together, the available data establish a link between the structural features of molecular interactions of CP molecules and the physical properties of helical virions ranging from rigidity to flexibility. Overall, the structure of helical plant viruses is significantly more labile than previously thought, often allowing structural transitions, remodelling and the existence of alternative structural forms of virions. These properties of virions are believed to be functionally significant at certain stages of the viral life cycle, such as during translational activation and cell-to-cell transport. In this review, we discuss structural and functional features of filamentous and rod-shaped virions, highlight their shared features and differences, and lay emphasis on the relationships between the molecular structure of viral capsids and their properties including virion shape, lability and capability of structural remodelling.