Investigation by Atomic Force Microscopy of the Structure of Ty3 Retrotransposon Particles

Abstract

Ty3, a member of the Metaviridiae family of long-terminal-repeat retrotransposons found in Saccharomyces cerevisiae, encodes homologs of retroviral Gag and Gag-Pol proteins, which, together with genomic RNA, assemble into virus-like particles (VLPs) that undergo processing and reverse transcription. The Ty3 structural proteins, capsid and nucleocapsid, contain major homology and nucleocapsid motifs similar to retrovirus capsid and nucleocapsid proteins, but Ty3 lacks a matrix-like structural domain amino terminal to capsid. Mass spectrometry analysis of Ty3 Gag3 processing products defined an acetylated Ser residue as the amino terminus of Gag3/p34, p27, and CA/p24 species and supported a model where p34 and p27 occur in phosphorylated forms. Using atomic force microscopy, VLPs were imaged from cells producing wild-type and protease and reverse transcriptase mutant Ty3. Wild-type VLPs were found to have a broad range of diameters, but the majority, if not all of the particles, exhibited arrangements of capsomeres on their surfaces which were consistent with icosahedral symmetry. Wild-type particles were in the range of 25 to 52 nm in diameter, with particles in the 42-to 52-nm diameter range consistent with T‫7؍‬ symmetry. Both classes of mutant VLPs fell into a narrower range of 44 to 53 nm in diameter and appeared to be consistent with T‫7؍‬ icosahedral symmetry. The smaller particles in the wild-type population likely correspond to VLPs that have progressed to reverse transcription or later stages, which do not occur in the protease and reverse transcriptase mutants. Ty3 VLPs did not undergo major external rearrangements during proteolytic maturation.This study was undertaken to investigate the virus-like particle (VLP) structure of the long-terminal-repeat (LTR) retrovirus like element Ty3 from Saccharomyces cerevisiae (75). The retrotransposon VLP is analogous to the nonenveloped core particle of retroviruses. Determination of detailed structures of retroviral cores is complicated, because cores are enveloped and heterogeneous and mature particles of many retroviruses are relatively fragile (20,79). It was of interest to determine whether the smaller size of the Ty3 structural domain would be reflected in a simplification of particle morphology. Retroviral cores are initially assembled from Gag together with lesser amounts of Gag-Pol to form spherical, immature cores. Naked (nonenveloped) cores range in average diameter from 79 nm for Rous sarcoma virus (RSV) (38) to 96 nm for murine leukemia virus (MLV) (86).

Atomic Force Microscopy Investigation of Viruses

“…A counter example was provided by the rod-shaped, helical tobacco mosaic virus RNA which appeared initially as a thread, a completely extended molecule lacking any secondary structure. With time, it began forming local secondary structural elements and eventually condensed into forms similar to those seen for the RNA from the icosahedral viruses (41). One conclusion of the study was that the single strands of RNA spontaneously condensed as linear arrangements of stem-loop substructures following synthesis, the condensed RNA bound coat protein to it, and the two cooperatively coalesced into the completed particle.…”

“…AFM investigations have been conducted on RNA extracted by phenol from a series of small icosahedral viruses, and from tobacco mosaic virus, the classical rod-shaped, helical virus (41). The spherical viruses included poliovirus, STMV, TYMV, and brome mosaic virus.…”

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

“…5d), it was deduced that Ty3 retrotransposon particles seen in Fig. 5c existed as icosahedra having triangulation numbers 3, 4, and 7 (23,31).…”

“…Instead, only an outer lipid membrane was visible. A study of Ty3 retrotransposon having a defective protease gene mutant and reverse transcriptase (RT) mutant Ty3 particles was illuminating in a different way (31). While wild-type virus appeared as T ϭ 3, 4, and 7 icosahedral particles, the RT and Pr mutants appeared to be exclusively T ϭ 7 particles.…”

Identification of DNA and RNA from retroviruses using ribonuclease A

“…In the course of the studies on retroviruses and retrotransposons, which are basically intracellular retroviruses (Kuznetsov et al 2005a, Sandmeyer et al 2002, we could, in general, recognize and discriminate RNA and DNA by their unique appearances, even at modest resolutions. In some cases, however, identification was ambiguous, and we felt it necessary to find some complementary approach to discrimination.…”