Identification of DNA and RNA from retroviruses using ribonuclease A

Kuznetsov, Yuri G.; McPherson, Alexander

doi:10.1002/sca.4950280506

Cited by 15 publications

(13 citation statements)

References 14 publications

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“…A method was devised for additional identification based on exposure of the nucleic acid to high concentrations of bovine RNase A (27). One example of results from that study is shown in Fig.…”

Section: Visualization Of Viral Rna and Dnamentioning

confidence: 99%

Atomic Force Microscopy in Imaging of Viruses and Virus-Infected Cells

Kuznetsov

McPherson

2011

Microbiol Mol Biol Rev

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125

101

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SUMMARY Atomic force microscopy (AFM) can visualize almost everything pertinent to structural virology and at resolutions that approach those for electron microscopy (EM). Membranes have been identified, RNA and DNA have been visualized, and large protein assemblies have been resolved into component substructures. Capsids of icosahedral viruses and the icosahedral capsids of enveloped viruses have been seen at high resolution, in some cases sufficiently high to deduce the arrangement of proteins in the capsomeres as well as the triangulation number ( T ). Viruses have been recorded budding from infected cells and suffering the consequences of a variety of stresses. Mutant viruses have been examined and phenotypes described. Unusual structural features have appeared, and the unexpectedly great amount of structural nonconformity within populations of particles has been documented. Samples may be imaged in air or in fluids (including culture medium or buffer), in situ on cell surfaces, or after histological procedures. AFM is nonintrusive and nondestructive, and it can be applied to soft biological samples, particularly when the tapping mode is employed. In principle, only a single cell or virion need be imaged to learn of its structure, though normally images of as many as is practical are collected. While lateral resolution, limited by the width of the cantilever tip, is a few nanometers, height resolution is exceptional, at approximately 0.5 nm. AFM produces three-dimensional, topological images that accurately depict the surface features of the virus or cell under study. The images resemble common light photographic images and require little interpretation. The structures of viruses observed by AFM are consistent with models derived by X-ray crystallography and cryo-EM.

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Section: Visualization Of Viral Rna and Dnamentioning

confidence: 99%

Atomic Force Microscopy in Imaging of Viruses and Virus-Infected Cells

Kuznetsov

McPherson

2011

Microbiol Mol Biol Rev

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125

101

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“…Sometimes, however, the distinction is not entirely clear and further evidence may be needed to show whether a filament, strand, or complex is DNA or RNA. A method was devised for additional identification based on exposure of the nucleic acid to high concentrations of bovine RNase A (42). RNA, naturally, was hydrolyzed to small pieces by RNase A and left only fragments on the substrate which corresponded to protected stem-loops.…”

Section: Imaging Of Nucleic Acids Of Virusesmentioning

confidence: 99%

Atomic Force Microscopy Investigation of Viruses

McPherson¹,

Kuznetsov²

2011

Methods in Molecular Biology

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Atomic force microscopy (AFM) has proven to be a valuable approach to delineate the architectures and detailed structural features of a wide variety of viruses. These have ranged from small plant satellite viruses of only 17 nm to the giant mimivirus of 750 nm diameter, and they have included diverse morphologies such as those represented by HIV, icosahedral particles, vaccinia, and bacteriophages. Because it is a surface technique, it provides images and information that are distinct from those obtained by electron microscopy, and in some cases, at even higher resolution. By enzymatic and chemical dissection of virions, internal structures can be revealed, as well as DNA and RNA. The method is relatively rapid and can be carried out on both fixed and unfixed samples in either air or fluids, including culture media. It is nondestructive and even non-perturbing. It can be applied to individual isolated virus, as well as to infected cells. AFM is still in its early development and holds great promise for further investigation of biological systems at the nanometer scale.Key words: Imaging, Nanoscale, Structure, Infection, Nucleic acids, Icosahedra A direct imaging technology that promises to have a significant impact on structural biology, and which is, in most ways, complementary to X-ray diffraction and electron microscopy, the classical approaches, is atomic force microscopy (AFM) (1-3). An immediate advantage of AFM is that it is based on relatively simple physical principles, unlike X-ray crystallography, and the instruments are mechanically and electronically rather straightforward, unlike electron microscopy. Unlike both of the other technologies, AFM is fairly inexpensive to institute and apply, even to biological specimens. The acuity and investigative size range of the AFM have proven to be quite remarkable and it is now permitting researchers new access to virus structure and the effects of viruses on organisms. Indeed, it has allowed us to

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“…AFM analysis was carried out using a Nanoscope III multimode instrument (Veeco Instruments, Santa Barbara, CA). Imaging procedures were fundamentally the same as described for previous investigations of viruses (Kuznetsov et al, 2001(Kuznetsov et al, , 2002(Kuznetsov et al, , 2003; and RNA (Kuznetsov et al, 2005b;Kuznetsov and McPherson, 2006). For scanning in air, silicon tips were employed.…”

Section: Methodsmentioning

confidence: 99%