Ty1 Virus-like Particles Research Articles

Gag-Specific Immune Responses after Immunization with pl7/p24:Ty Virus-Like Particles in HIV Type 1-Seropositive Individuals

Gag-specific immune responses and changes in HIV-1 RNA levels were evaluated in eight HIV-1-infected persons, in order to assess the immunotherapeutic potential HIV-1 p17/p24: Ty virus-like particles (p24-VLP). All treated subjects showed transient and dose-dependent proliferative responses to the Ty-VLP carrier (stimulation index [SI], 2.0-119.5). Three of four individuals who received either 500 or 1,000 micrograms of p24-VLP also showed proliferative responses to p17 or p24 (SI, 2.0-15.7). In 2 subjects who were treated with either 500 or 1,000 micrograms of p24-VLP, enhanced Gag-specific CTL precursor (CTLp) frequencies were observed after immunization (10- to 14-fold). Both subjects had low baseline Gag-specific CTL activity (< 25 cTLp/10(6) PBMCs). In the other participants studied no significant boosting of preexisting Gag-specific CTL responses was observed. Short-term elevation of HIV-1 RNA levels at weeks 2 and 4 was observed in two subjects treated with the highest dose of p24-VLP. However, HIV-1 RNA levels at week 24 did not significantly differ from those found in the placebo group. In conclusion, p24-VLP induced marginal Gag-specific immune responses in limited numbers of HIV-1-seropositive individuals, with some showing transient elevation of HIV-1 viral load. Further studies are needed to establish potential clinical effects of these observations.

Prediction of murine MHC class I epitopes in a major house dust mite allergen and induction of T1-type CD8+ T cell responses.

The group I (Der p 1) allergen of Dermatophagoides pteronyssinus (house dust mite, HDM) contains several T helper (Th) epitopes recognized by C57BL/6 mice, with the peptide (111-139) containing a dominant MHC class II-restricted epitope (113-127). Since CD8+ T cells are thought to play a role in the regulation of allergic disease, we examined the Der p 1 sequence for potential MHC class I-binding motifs and observed that residues 111-119 (FGISNYCQI) contain motifs for H-2Db and Kb. Furthermore, immunization of C57BL/6 mice with unadjuvanted Ty virus-like particles (VLP) carrying Der p 1 (111-139), a method known to induce murine cytotoxic T lymphocyte (CTL) responses, primed Der p 1 (111-119)-specific Db-restricted CTL which produce high levels of IFN-gamma and low levels of IL-5 and IL-6 in vitro (T1-type CTL). VLP carrying the minimal epitope (FGISNYCQI) also induced a CTL response following immunization without adjuvant by various routes. Der p 1 (111-139)-VLP adjuvanted with alum did not prime CTL in C57BL/6 mice but were found to prime Th1-type CD4+ T cells that recognize the overlapping peptide (113-127) and native protein. The ability to successfully predict allergen-specific CD8+ T cell epitopes and prime CD8+ and/or CD4+ T cell responses provides an opportunity to dissect the relative roles of these T cells in the regulation of allergic responses and may offer therapeutic potential for reprogramming Th2-type allergic responses.

Mapping the multimerization domains of the Gag protein of yeast retrotransposon Ty1.

The two-hybrid system was used to define regions of the Ty1 Gag protein responsible for multimerization. Gag truncations lacking the first 146 or the last 97 amino acids (Gag is 440 amino acids in length) interact. A severely C-terminally truncated molecule (lacking the last 207 amino acids) was the smallest truncation to interact, suggesting that some protein-protein interactions between Gag molecules are mediated through the first 233 amino acids. However, an internal deletion of amino acids 147 to 233 does not abolish Gag-Gag interaction, indicating that more than one region can mediate Gag interaction. Surprisingly, we found that a truncation lacking the last 97 amino acids interacts with itself but not with full-length Gag. This is apparently due to an artifact of the two-hybrid assay, since these same molecules coassemble with wild-type Gag into Ty1 virus-like particles.

Structural determinants within the subunit protein of Ty1 virus-like particles.

The Ty virus-like particles (VLPs) are functionally analogous to retroviral particles. They package the enzymes and the RNA necessary for retrotransposition, and mediate the integration of the reverse-transcription product into the genome of the host cell. Here we map three structural determinants of particle assembly in the subunit protein. We have also identified key residues in these regions that seem to be involved in subunit interaction and particle morphology. In particular, two point mutations in putative amphipathic helices have remarkable effects on VLP morphology, increasing the diameter as much as eightfold.

Ty3 integrase mutants defective in reverse transcription or 3'-end processing of extrachromosomal Ty3 DNA

Ty3, a retroviruslike element in Saccharomyces cerevisiae, encodes an integrase (IN) which is essential for position-specific transposition. The Ty3 integrase contains the highly conserved His-Xaa(3-7)-His-Xaa(23-32)-Cys-Xaa(2)-Cys and Asp, Asp-Xaa(35)-Glu [D,D(35)E] motifs found in retroviral integrases. Mutations were introduced into the coding region for the Ty3 integrase to determine the effects in vivo of changes in conserved residues of the putative catalytic triad D,D(35)E and the nonconserved carboxyl-terminal region. Ty3 viruslike particles were found to be associated with significant amounts of linear DNA of the approximate size expected for a full-length reverse transcription product and with plus-strand strong-stop DNA. The full-length, preintegrative DNA has at each 3' end 2 bp that are removed prior to or during integration. Such 3'-end processing has not been observed for other retroviruslike elements. A mutation at either D-225 or E-261 of the Ty3 integrase blocked transposition and prevented processing of the 3' ends of Ty3 DNA in vivo, suggesting that the D,D(35)E region is part of the catalytic domain of Ty3 IN. Carboxyl-terminal deletions of integrase caused a dramatic reduction in the amount of Ty3 DNA in vivo and a decrease in reverse transcriptase activity in vitro but did not affect the apparent size or amount of the 55-kDa reverse transcriptase in viruslike particles. The 115-kDa viruslike particle protein, previously shown to react with antibodies to Ty3 integrase, was shown to be a reverse transcriptase-IN fusion protein. These results are consistent with a role for the integrase domain either in proper folding of reverse transcriptase or as part of a heterodimeric reverse transcriptase molecule.

Induction of single and dual cytotoxic T‐lymphocyte responses to viral proteins in mice using recombinant hybrid Ty–virus‐like particles

The induction of cytotoxic T-lymphocyte (CTL) responses to viral proteins is thought to be an essential component of protective immunity against viral infections. Methods for generating such responses in a reproducible manner would be of great value in vaccine development. We demonstrate here that the recombinant antigen-presentation system based on the yeast transposon (Ty) particle-forming p1 protein is a potent means of inducing CTL responses to a variety of viral CTL epitopes, including influenza virus nucleoprotein (two epitopes), Sendai virus and vesicular stomatitis virus nucleoproteins, and the V3 loop of human immunodeficiency virus type-1 (HIV-1) gp120. CTL were primed by hybrid Ty-virus-like particles (VLP) carrying the minimal epitope or as much as 19,000 MW of protein. Ty-VLP carrying two different epitopes (dual-epitope Ty-VLP) were capable of priming CTL responses in two different strains of mice or against two epitopes in the same individual. Furthermore, co-administration of a mixture of two different Ty-VLP carrying single epitopes could induce responses to both epitopes in the same individual. Ty-VLP appear to represent a reproducible and flexible system for inducing CTL responses in mice, and warrant further evaluation in primates.

Application of Ty1 for cloned gene insertion: amplification of a large regulated expression cassette in Saccharomyces cerevisiae

A large cassette, 4.6 x 10(3) bases (4.6 kb) in length, containing an inducible expression system (the yeast CUP1 promoter fused to the Escherichia coli lacZ structural gene) and a bacterial neomycin-resistance gene (neo) has been cloned into the noncoding region of a GAL1-regulated Ty1 retrotransposon. Galactose was used to induce retrotransposition in Saccharomyces cerevisiae, and cells containing integrations were selected by resistance to the aminoglycoside G418. Integrations of neo and CUP1p-lacZ were verified, and beta-galactosidase activity was confirmed. Analysis via Southern blots demonstrated integrations at various chromosomal locations, and the number of insertions obtained ranged from one to five after three rounds of induction. Therefore, the packaging limit of Ty1 virus-like particles for RNA is at least 10.3 kb and Ty1 can transpose foreign genes as large as 4.6 kb, demonstrating the practical application of Ty1 for the insertion of large regulated expression cassettes.

Immunogenicity of the yeast recombinant [formula omitted]:Ty virus-like particles (p24-VLP) in healthy volunteers

In this Phase I study, immunisation with the yeast-derived p24 virus-like particles Ty p24-VLP (3 × 100 or 3 × 500 μg subcutaneously) in 16 healthy male subjects elicited p24 antibody responses in 4 of 16 (25%) subjects. After a fourth, intramuscular, immunisation (500 μg), p24 antibody responses were detected in 11 of 15 (70%) subjects. In addition to p24 antibody responses, T cell proliferative responses were also observed, although no HLA restricted p24-specific cytotoxic T cell responses were detected. The results demonstrate that Ty p24-VLP is immunogenic and well-tolerated in healthy male subjects.

Requirement of RNA Polymerase III Transcription Factors for in Vitro Position-Specific Integration of a Retroviruslike Element

The yeast retroviruslike element Ty3 inserts at the transcription initiation sites of genes transcribed by RNA polymerase III (Pol III). An in vitro integration assay was developed with the use of Ty3 viruslike particles and a modified SUP2 tyrosine transfer RNA (tRNA(Tyr)) gene target. Integration was position-specific and required Ty3 integrase, Pol III transcription factor (TF) IIIB-, TFIIIC-, and Pol III-containing fractions showed that TFIIIB and TFIIIC, together, were sufficient for position-specific Ty3 integration, but not for transcription. This report demonstrates that in vitro integration of a retroelement can be targeted by cellular proteins.

Transposition of the yeast retroviruslike element Ty3 is dependent on the cell cycle.

Host cell cycle genes provide important functions to retroviruses and retroviruslike elements. To define some of these functions, the cell cycle dependence of transposition of the yeast retroviruslike element Ty3 was examined. Ty3 is unique among retroviruslike elements because of the specificity of its integration, which occurs upstream of genes transcribed by RNA polymerase III. A physical assay for Ty3 transposition which takes advantage of this position-specific integration was developed. The assay uses PCR to amplify a product of Ty3 integration into a target plasmid that carries a modified tRNA gene. By using the GAL1 upstream activating sequence to regulate expression of Ty3, transposition was detected within one generation of cell growth after Ty3 transcription was initiated. This physical assay was used to show that Ty3 did not transpose when yeast cells were arrested in G1 during treatment with the mating pheromone alpha-factor. The restriction of transposition was not due to changes in transcription of either Ty3 or tRNA genes or to aspects of the mating pheromone response unrelated to cell cycle arrest. The block of the Ty3 life cycle was reversed when cells were released from G1 arrest. Examination of Ty3 intermediates during G1 arrest indicated that Ty3 viruslike particles were present but that reverse transcription of the Ty3 genomic RNA into double-stranded DNA had not occurred. In G1, the Ty3 life cycle is blocked after particle assembly but before the completion of reverse transcription.

Transposition of the yeast retroviruslike element Ty3 is dependent on the cell cycle.

Host cell cycle genes provide important functions to retroviruses and retroviruslike elements. To define some of these functions, the cell cycle dependence of transposition of the yeast retroviruslike element Ty3 was examined. Ty3 is unique among retroviruslike elements because of the specificity of its integration, which occurs upstream of genes transcribed by RNA polymerase III. A physical assay for Ty3 transposition which takes advantage of this position-specific integration was developed. The assay uses PCR to amplify a product of Ty3 integration into a target plasmid that carries a modified tRNA gene. By using the GAL1 upstream activating sequence to regulate expression of Ty3, transposition was detected within one generation of cell growth after Ty3 transcription was initiated. This physical assay was used to show that Ty3 did not transpose when yeast cells were arrested in G1 during treatment with the mating pheromone alpha-factor. The restriction of transposition was not due to changes in transcription of either Ty3 or tRNA genes or to aspects of the mating pheromone response unrelated to cell cycle arrest. The block of the Ty3 life cycle was reversed when cells were released from G1 arrest. Examination of Ty3 intermediates during G1 arrest indicated that Ty3 viruslike particles were present but that reverse transcription of the Ty3 genomic RNA into double-stranded DNA had not occurred. In G1, the Ty3 life cycle is blocked after particle assembly but before the completion of reverse transcription.

Targeted lymph node immunization with simian immunodeficiency virus p27 antigen to elicit genital, rectal, and urinary immune responses in nonhuman primates.

A s.c. route of immunization was developed in non-human primates, which targets the genitourinary-rectal associated lymphoid tissue. A vaccine consisting of rSIV gag p27, expressed as hybrid Ty virus-like particles (p27: Ty-VLP) was administered in the proximity of the internal iliac lymph nodes. Secretory IgA and IgG Abs to the p27 Ag were elicited in the vaginal, male urethral, rectal and seminal fluids, urine and serum. Two or more immunodominant B cell epitopes were identified within peptides 51-90 and 121-170 of the sequence of p27, using serum or biliary IgA and IgG Abs. CD4+ T cell proliferative responses to p27 were elicited predominantly in the targeted internal iliac, as well as the inferior mesenteric lymph nodes and the spleen, but not in the unrelated lymph nodes. These cells were then studied for helper function in p27 specific B cell Ab synthesis. Specific IgA and IgG Abs were detected in the same lymphoid tissues as those that displayed proliferative responses. However, cross-over reconstitution experiments between splenic and iliac lymph node B and CD4+ T cells suggest that the iliac B cells are essential for specific IgA Ab synthesis, whereas splenic B cells preferentially synthesize IgG Ab. The targeted lymph node (TLN) route of immunization gave comparable B cell, proliferative T cell, and Th cell responses to the vaginal, male genitourinary, and rectal mucosal routes, which were augmented by oral immunization. However, the TLN route induced urinary and seminal fluid sIgA and IgG Abs in addition to genital and rectal Abs. Generating secretory IgA and IgG Abs at the mucosal surfaces, and T and B cell immunity in the regional draining lymph nodes, spleen and circulation by TLN immunization may prevent transmission of virus through the mucosa, dissemination of the virus, and the formation of a latent reservoir of infection.

Expression and partial purification of enzymaticallyactive recombinant Ty1 integrase in Saccharomyces cerevisiae.

Integration of the Saccharomyces cerevisiae retrotransposon Ty1 into the genome requires Ty1 integrase (IN). Apparent functions of Ty1 IN are target-site determination, cleavage, and joining of donor strands. To further study the mechanism of Ty1 integration, an IN expression plasmid has been constructed for use in yeast. The recombinant IN coding sequence differs from mature Ty1 IN associated with Ty1 virus-like particles only in that it has several additional N-terminal amino acid codons. Inclusion of a polyhistidine tag facilitates purification of recombinant IN by metal chelate chromatography. Recombinant Ty1 IN is active in an in vitro assay with short double-stranded oligonucleotide substrates and has biochemical properties similar to those observed with Ty1 virus-like particles. The full-length Ty1 IN produced in yeast should be useful for further biochemical, genetic, and structural analyses of Ty1 integration and for comparative analyses with retroviral IN proteins.

Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis.

We have developed efficient methods for creating artificial transposons and inserting these transposons into plasmid targets in vitro, primarily for the purpose of DNA mapping and sequencing. A novel plasmid has been engineered to convert virtually any DNA sequence, or combination of sequences, into an artificial transposon; hence, custom transposons containing any desired feature can be easily designed and constructed. Such transposons are then efficiently inserted into plasmid targets, in vitro, using the integrase activity present in yeast Ty1 virus-like particles. A single in vitro integration reaction, which resembles a simple restriction digestion in the complexity of the reaction, gives rise to thousands of recoverable insertion events within DNA target molecules; this frequency approaches one insertion per phosphodiester bond in typical plasmids. Importantly, transposon insertions are recovered from all regions of DNA inserts carried on plasmid targets, indicating that integration is a random or nearly-random process. Because of its versatility, this technology offers a generalized method of generating recombinant DNA molecules of a desired structure. We have adapted this system for DNA sequencing by developing a customized artificial transposon to insert new primer binding sites into internal regions of DNA inserts carried on cloning vectors. Transposon insertions have been generated throughout several different yeast and human DNA inserts carried on plasmids, allowing the efficient recovery of sequence information from these inserts. Our results demonstrate the overall utility of this method for both small and large-scale DNA sequencing, as well as general DNA restructuring, and indicate that it could be adapted for use with a number of additional applications including functional genetic analysis.

Immunization of human HIV-seronegative volunteers with recombinant p17/p24

To evaluate the immune response to HIV-1 p24 generated in vivo by p17/p24:Ty virus-like particles (p17/p24:Ty-VLP) by examining the lymphoproliferative and antibody (Ab) responses to HIV-1 p24, as well as Gag-specific cytotoxic T lymphocytes (CTL), in HIV-seronegative volunteers immunized with hybrid p17/p24:Ty-VLP. Sixteen HIV-seronegative volunteers were immunized with p17/p24:Ty-VLP at two dose levels (100 or 500 micrograms) and monitored for the following 48 weeks for production of anti-p24 and anti-p17 Ab, in vitro lymphoproliferative responses to HIV-1 p24 and p17, and in vitro CTL responses to HIV-1 Gag. Twelve out of the 16 volunteers had significant p24-specific proliferative responses, with volunteers on the higher dose schedule exhibiting earlier proliferative responses than those on the lower dose schedule. Proliferative responses in both volunteer groups were similar in overall magnitude but appeared at different times during the immunization schedule. Anti-p24 Ab were detected in six out of the nine individuals in the lower dose group and in five out of the seven in the higher dose group. There was a good correlation between the presence of p24-specific Ab and the detection of lymphoproliferative responses to the p24 protein in peripheral blood mononuclear cells isolated from the same individuals. Anti-p17 Ab were detected in five volunteers. No Gag-specific CTL responses were detected. We conclude that hybrid HIV-1 p17/p24:Ty-VLP are capable of inducing both cellular and humoral immunity to HIV-1 Gag p17 and p24 components and are worthy of further study as a potential HIV immunotherapeutic.

T- and B-cell functions and epitope expression in nonhuman primates immunized with simian immunodeficiency virus antigen by the rectal route.

Transmission of human immunodeficiency virus (HIV) in North America and Europe occurs most commonly through the rectal mucosa during homosexual intercourse. The simian immunodeficiency virus (SIV) macaque model has been used to investigate rectal immunization. The vaccine used was a recombinant SIV gag p27 expressed as hybrid Ty virus-like particles (Ty-VLP). Sequential ororectal (OR) mucosal immunization was compared with i.m. immunization. Whereas both routes of immunization induced serum IgA and IgG p27 antibodies, only OR immunization induced rectal secretory IgA antibodies. Specific CD4+ T-cell proliferative responses to stimulation with p27 were found after i.m. immunization only in the blood and spleen, but after OR immunization they were found in the internal iliac and inferior mesenteric lymph nodes in addition to the blood and spleen. T-cell epitope mapping of the proliferative responses of short-term cell lines (STCLs) grown from peripheral blood or lymphoid cells revealed a major epitope within the polypeptide 121-150 after either route of immunization. Two minor T-cell epitopes were found within peptide 41-80 in STCLs from splenic and circulating cells. B-cell epitope mapping of serum or biliary IgA and IgG antibodies revealed two overlapping or adjacent immunodominant epitopes to the T-cell epitopes within the polypeptides 121-170 and 51-90. The results suggest that rectal augmented by oral immunization with a recombinant particulate antigen in nonhuman primates elicits secretory IgA and to a lesser extent IgG responses in the draining lymph nodes and the rectal mucosa, whereas systemic immunization targets predominantly splenic and circulating T- and B-cell responses. These findings may have important implications in the strategy of designing vaccines in prevention of homosexual transmission of HIV infection.

Induction of HIV-specific cytotoxic T lymphocytes in vivo with hybrid HIV-1 V3:Ty-virus-like particles.

Abstract In general, it has proven difficult to induce CTL responses using simple proteins or peptides without resorting to specialized adjuvants. In this study we show that particulate polymeric Ag in the form of hybrid Ty virus-like particles carrying the V3 region of HIV-1 gp120/160 envelope protein (V3:Ty-VLP) induce V3-specific CTL in BALB/c mice in the absence of adjuvant or lipid vehicle. In vitro restimulation of splenocytes with V3 peptide was necessary in order to generate effector CTL. Th cell activation was not required for this in vitro restimulation phase. The CTL induced by the V3:Ty-VLP were CD8+ve, H-2d-restricted, and HIV-1 isolate-specific (IIIB or MN). Co-administration of IIIB V3:Ty-VLP and MN V3:Ty-VLP primed both IIIB and MN V3-specific CTL. However, only IIIB V3-specific CTL were primed by hybrid Ty-VLP carrying IIIB, MN, and RF V3 loop sequences on the same particle indicating that there is intra- but not intermolecular competition between CTL epitopes. In direct comparisons, V3:Ty-VLP were substantially more potent than rgp120. Rgp160 and a 40mer IIIB V3 peptide both failed to prime V3-specific CTL. These data suggest that the particulate nature of hybrid Ty-VLP facilitates uptake into APC with subsequent access to the MHC class I processing pathway and that they may be useful vaccine vehicles for inducing cytolytic immunity against HIV-1 and other intracellular pathogens.

A very poorly expressed tRNA(Ser) is highly concentrated together with replication primer initiator tRNA(Met) in the yeast Ty1 virus-like particles.

The analysis of the tRNAs associated to the virus-like particles produced by the Ty1 element revealed the specific packaging of three major tRNA species, in about equal amounts: the replication primer initiator tRNA(Met), the tRNA(Ser)AGA and a tRNA undetected until now as an expressed species in yeast. The latter tRNA is coded by the already described tDNA(Ser)GCT. This tRNA is enriched more than 150 fold in the particles as compared to its content in total cellular tRNA where it represents less than 0.1% (initiator tRNA(Met) and tRNA(Ser)AGA being 11 and 4 fold enriched respectively). This tRNA is the only species coded by the tDNA(Ser)GCT gene which is found in three copies per genome since no other corresponding expressed tRNA could be detected. This gene is thus very poorly expressed. The high concentration of tRNA(Ser)GCU in the particles compared to its very low cellular content led us to consider its possible implication in Ty specific processes.

Initiator methionine tRNA is essential for Ty1 transposition in yeast.

The yeast retrotransposon Ty1 transposes through an RNA intermediate by a mechanism similar to that of retroviral reverse transcription and integration. Ty1 RNA contains a putative minus strand primer binding site (-PBS) that is complementary to the 3' acceptor stem of the initiator methionine tRNA (tRNA(iMet)). Here we demonstrate that the tRNA(iMet) is used as a primer for Ty1 reverse transcription. Mutations in the Ty1 element that alter 5 of 10 nucleotides that are complementary to the tRNA(iMet) abolish Ty1 transposition, even though they are silent with regard to Ty1 protein coding. We have constructed a yeast strain lacking wild-type tRNA(iMet) that is dependent on a mutant derivative of tRNA(iMet) that has an altered acceptor stem sequence, engineered to restore homology with the Ty1 -PBS mutant. The compensatory mutations made in the tRNA(iMet) alleviate the transposition defect of the Ty1 -PBS mutant. The mutant and wild-type tRNA(iMet) are enriched within Ty1 virus-like particles irrespective of complementarity to the Ty1 -PBS. Thus, complementarity between the Ty1 -PBS and tRNA(iMet) is essential for transposition but is not necessary for packaging of the tRNA inside virus-like particles.

Ty3 GAG3 and POL3 genes encode the components of intracellular particles.

Ty3 is a Saccharomyces cerevisiae retrotransposon that integrates near the transcription initiation sites of polymerase III-transcribed genes. It is distinct from the copialike Ty1 and Ty2 retrotransposons of S. cerevisiae in both the sequences of encoded proteins and gene order. It is a member of the gypsylike family of retrotransposons which resemble animal retroviruses. This study was undertaken to investigate the nucleocapsid particle of a transpositionally active gypsylike retrotransposon. Characterization of extracts from cells in which Ty3 expression was induced showed the presence of Ty3 nucleoprotein complexes, or viruslike particles, that migrated on linear sucrose gradients with a size of 156S. These particles are composed of Ty3 RNA, full-length, linear DNA, and proteins. In this study, antibodies raised against peptides predicted from the Ty3 sequence were used to identify Ty3-encoded proteins. These include the capsid (26 kDa), nucleocapsid (9 kDa), and reverse transcriptase (55 kDa) proteins. Ty3 integrase proteins of 61 and 58 kDa were identified previously (L. J. Hansen and S. B. Sandmeyer, J. Virol. 64:2599-2607, 1990). Reverse transcriptase activity associated with the particles was measured by using exogenous and endogenous primer-templates. Immunofluorescence studies of cells overexpressing Ty3 revealed cytoplasmic clusters of immunoreactive proteins. Transmission electron microscopy showed that Ty3 viruslike particles are about 50 nm in diameter. Thus, despite the unusual position specificity of Ty3 upstream of tRNA-coding regions, aspects of the Ty3 life cycle are fundamentally similar to those of retroviruses.