Department of Biological Chemistry

School of Medicine

University of California, Irvine

Irvine, CA 92625

SANDMEYER LABORATORY: RETROVIRUS MODELS IN YEAST

Retroviruslike lifecycle:  transcription is followed by export of RNA which is translated into Ty3 proteins and packaged into viruslike particles (VLPs) composed of the encoded protein.  Reverse transcription occurs in association with the VLPs.  The VLP is probably substantially remodeled in order to translocate into the nucleus where the preintegration complex integrates at the initiation sites of genes transcribed by RNA Pol III.  Processes of assembly, nuclear entry, and integration targeting are not well understood for Ty3 or for most retroviruses.

Ty3 VLP structure

Ty3 position specificity

Ty3 retrotransposition and host genes

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Retroelements comprise approximately half of the human genome and we are only beginning to understand the role that these elements play in human disease and development and in genome evolution.   The Sandmeyer laboratory discovered and now studies the budding yeast retroviruslike element Ty3 as a retrovirus model in a simple host organism.  Retroviruses are the causative agent of acquired immunodeficiency disease syndrome and are the basis of some gene therapy vectors.  

Ty3 is 5.4 kb in length and is comprised of 340 bp long terminal repeats (LTRs) flanking an internal coding domain of two overlapping open reading frames, GAG3 and POL3.  GAG3 encodes the structural proteins capsid (CA) and nucleocapsid (NC), and POL3, which is translated via a translational frameshift from GAG3, encodes the catalytic proteins protease (PR), reverse transcriptase (RT) and integrase (IN).  Thus, the Ty3 genome is organized similarly to a simple retrovirus genome and with the exceptions of matrix and envelope, encodes proteins with functions similar to those in common among the simple retroviruses. 

Ty3 VLP structure

Expression of Ty3 results in production of Ty3 proteins which assemble together with genomic RNA into viruslike particles (VLPs).  Particle formation is concomitant with proteolytic processing which is required for reverse transcription.  Full-length cDNA is observed by about 4 h by Southern blot analysis and integrated elements are reliably detectable by 6 h by amplification using a polymerase chain reaction. The Sandmeyer laboratory has three major areas of interest: 1) structural analysis of Ty3 particle morphogenesis; 2) the mechanism of Ty3 integration site targeting; and 3) the role of host factors in the Ty3 lifecycle.  The Sandmeyer laboratory has collaborations in progress with mass spectrometry, cryoelectron microscopy, crystallography, and atomic force microscopy laboratories in order to characterize the Ty3 particle at different points in the morphogenetic pathway.  Major goals include documenting the internal structural changes that accompany protein processing, identification and localization of modifications that may have regulatory significance, and characterization of the form of the particle as it undergoes reverse transcription and integration.  In the past year, atomic force microscopic characterization of the Ty3 particle was performed in collaboration with Y. Kuznetsov in the A. McPherson laboratory (UCI)(see images in left panel). 

Ty3 wild type and PR and RT mutant particles were characterized using atomic force microscopy (left panel).  Ty3 particles are roughly 50 nm in diameter.  Surprisingly, PR processing did not affect the external structure as occurs for retroviruses.  Rather cDNA production appear to be associated with more heterogeneous forms of particles.  In addition, immature and processed VLPs were shown to have pentagonal elements of icosahedral symmetry, which have been hypothesized, but not directly demonstrated for the retrovirus core particle structure. It was also shown that expression of Gag3 alone is sufficient for VLP formation.  This work is described in Kuznetsov et al. (2005).  Because Ty3 VLPs are too large to transit the nuclear pore and one Ty3 nuclear localization sequence has been identified in IN.  It is presumed that extensive remodeling of this structure must occur prior to nuclear entry.

Ty3 position specificity

A matter of particular concern with respect to retrovirus-based gene therapy vectors is target site selection.  Retroviruses can not only disrupt expression or function of a gene, but can also activate expression by introduction of new promoter or enhancer sequences.  Although retroviruses are relatively promiscuous in their insertion patterns, recent reports indicate that they have virus class specific regional insertion biases.  In the case of the yeast elements, Ty1, 2 and 4 target the region upstream of tRNA genes. Ty5 targets heterochromatic regions.  Ty3 inserts specifically into the position of transcription initiation of genes transcribed by RNA polymerase III (e.g. tRNA genes, 5S genes, and U6 genes (see diagram where arrows above and below initiation site indicate point of Ty3 strand transfer).  In order to define the insertion site elements important for targeting, an in vitro system was developed in collaboration with E. P. Geiduschek and G. Kassavetis (UCSD) using the U6 gene, which is transcribed by RNA polymerase III, as a target and recombinant TFIIIB proteins.  Using this system, TFIIIB subunits TBP and Brf1 were shown to be the minimal factors required for position-specific Ty3 in vitro integration.  Although in vitro position-specific integration systems are available for prokaryotic elements, this is the first eukarytic transposable in vitro integration system that displays the native pattern of insertion specificity based on target site DNA binding factors.  This work is described in several papers including Kirchner et al. (1995), Connolly and Sandmeyer (1997), and Yieh et al. (2000, 2002). Current work in the laboratory indicates that the transcription factor Brf1 interacts directly with the Ty3 IN.  Although targeting of mutants that are disrupted for this interaction remains to be tested in vivo, these data suggest that Brf1 mediates position specificity.  In order to better understand the possible interactions between IN and Brf1, for which there is a DNA bound structure, the Sandmeyer laboratory is collaborating with the Baldi bioinformatics laboratory (UCI), which has expertise in molecular modeling.  A future goal of the laboratory is to apply the understanding of Ty3 targeted integration to the design of retroviral vectors with novel insertion specificity.

Ty3 retrotransposition and host genes

GOnet visualization of cluster of genes identified in Ty3 mutant screen.  Red nodes represent genes identified in Ty3 screen; blue nodes identify genes identified in Griffith et al. (Devine lab) Ty1 screen. For GOnet yeast interaction interactive database see http://contact5.ics.uci.edu/gonet/sgdindex/html.  GOnet was developed in collaboration with P. Baldi and his graduate student Y. Dou (UCI Information and Computer Sciences and Institute for Genomics and Bioinformatics).

The last area of interest is the role of host genes in the Ty3 lifecycle.  During the past review period, two genomewide screens were conducted and identified a total of about 180 genes that directly or indirectly affect the Ty3 lifecycle.  These results were reported by Aye et al. (2001, 2003, 2004) and Irwin et al. (2005).  Identified genes overlapped with genes previously identified in two independent searches for Ty1 host genes, which identified nonoverlapping sets of genes, and also included genes previously identified as affecting retrovirus replication.  Genes that affected Ty3 transposition included ones encoding factors involved in many cellular processes including transcription, RNA processing, vesicular trafficking, nuclear entry, and DNA maintenance.  Immunoblot analysis using antibodies against Ty3 proteins showed that some of the host mutants had reduced or elevated amounts of Ty3 proteins.  Interesting groups of nuclear pore proteins and DNA maintenance proteins were identified using the GOnet web-based interactive database (see above figure legend). Deletion of  genes encoding these factors resulted in elevated Ty3 cDNA and transposition, suggesting an intriguing link between nuclear entry, uncoating and cDNA production.  The contributions of host genes to specific stages of assembly and particle morphogenesis are being explored using the genetic and biochemical assays and other resources developed during previous research on the Ty3 element.

The laboratory is supported by grants from the National Institutes of Health, the National Science Foundation, and the University of California AIDS Researach Program.