Adeno-associated virus Rep proteins target DNA sequences to a unique locus in the human genome.

We have developed a system for site-specific DNA integration in human cells, mediated by the adeno-associated virus (AAV) Rep proteins. In its normal lysogenic cycle, AAV integrates at a site on human chromosome 19 termed AAVS1. We describe a rapid PCR assay for the detection of integration events at AAVS1 in whole populations of cells. Using this assay, we determined that the AAV Rep proteins, delivered in cis or trans, are required for integration at AAVS1. Only the large forms of the Rep protein, Rep78 and Rep68, promoted site-specific integration. The AAV inverted terminal repeats, present in cis, were not essential for integration at AAVS1, but in cells containing Rep, they increased the efficiency of integration. In the presence of the Rep proteins, the integration of a plasmid containing AAV inverted terminal repeats occurred at high frequency, such that clones containing the plasmid could be isolated without selection. In two of the five clones analyzed by fluorescence in situ hybridization, the plasmid DNA was integrated at AAVS1. In most of the clones, at least one copy of the entire plasmid was integrated in a tandem array. Detailed analysis of the integrated plasmid structure in one clone suggested a complex mechanism producing rearrangements of the flanking genomic DNA, similar to those observed with wild-type AAV.

Therapeutic effect of Gag-nuclease fusion protein on retrovirus-infected cell cultures.

Capsid-targeted viral inactivation is a novel protein-based strategy for the treatment of viral infections. Virus particles are inactivated by targeting toxic fusion proteins to virions, where they destroy viral components from within. We have fused Staphylococcus nuclease (SN) to the C-terminal end of Moloney murine leukemia virus Gag and demonstrated that expression of this fusion protein in chronically infected chicken embryo fibroblasts resulted in its incorporation into virions and subsequent inactivation of the virus particles by degradation of viral RNA. Release of particles incorporating Gag-SN fusion proteins into the extracellular milieu activates the nuclease and results in destruction of the virion from within. By comparing the effects of incorporated SN and SN*, an enzymatically inactive missense mutant form of SN, on the infectivity of virus particles, we have clearly demonstrated that nucleolytic activity is the antiviral mechanism. Expression of Gag-SN fusion proteins as a therapeutic agent causes a stable reduction of infectious titers by 20- to 60-fold. The antiviral effect of capsid-targeted viral inactivation in our model system, using both prophylactic and therapeutic approaches, suggests that a similar anti-human immunodeficiency virus strategy might be successful.

Targeting foreign proteins to human immunodeficiency virus particles via fusion with Vpr and Vpx.

The human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vpr and Vpx proteins are packaged into virions through virus type-specific interactions with the Gag polyprotein precursor. To examine whether HIV-1 Vpr (Vpr1) and HIV-2 Vpx (Vpx2) could be used to target foreign proteins to the HIV particle, their open reading frames were fused in frame with genes encoding the bacterial staphylococcal nuclease (SN), an enzymatically inactive mutant of SN (SN*), and chloramphenicol acetyltransferase (CAT). Transient expression in a T7-based vaccinia virus system demonstrated the synthesis of appropriately sized Vpr1-SN/SN* and Vpx2-SN/SN* fusion proteins which, when coexpressed with their cognate p55Gag protein, were efficiently incorporated into virus-like particles. Packaging of the fusion proteins was dependent on virus type-specific determinants, as previously seen with wild-type Vpr and Vpx proteins. Particle-associated Vpr1-SN and Vpx2-SN fusion proteins were enzymatically active, as determined by in vitro digestion of lambda phage DNA. To determine whether functional Vpr1 and Vpx2 fusion proteins could be targeted to HIV particles, the gene fusions were cloned into an HIV-2 long terminal repeat/Rev response element-regulated expression vector and cotransfected with wild-type HIV-1 and HIV-2 proviruses. Western blot (immunoblot) analysis of sucrose gradient-purified virions revealed that both Vpr1 and Vpx2 fusion proteins were efficiently packaged regardless of whether SN, SN*, or CAT was used as the C-terminal fusion partner. Moreover, the fusion proteins remained enzymatically active and were packaged in the presence of wild-type Vpr and Vpx proteins. Interestingly, virions also contained smaller proteins that reacted with antibodies specific for the accessory proteins as well as SN and CAT fusion partners. Since similar proteins were absent from Gag-derived virus-like particles and from virions propagated in the presence of an HIV protease inhibitor, they must represent cleavage products produced by the viral protease. Taken together, these results demonstrate that Vpr and Vpx can be used to target functional proteins, including potentially deleterious enzymes, to the human or simian immunodeficiency virus particle. These properties may be exploitable for studies of HIV particle assembly and maturation and for the development of novel antiviral strategies.

Targeting of a nuclease to murine leukemia virus capsids inhibits viral multiplication.

Capsid-targeted viral inactivation is an antiviral strategy in which toxic fusion proteins are targeted to virions, where they inhibit viral multiplication by destroying viral components. These fusion proteins consist of a virion structural protein moiety and an enzymatic moiety such as a nuclease. Such fusion proteins can severely inhibit transposition of yeast retrotransposon Ty1, an element whose transposition mechanistically resembles retroviral multiplication. We demonstrate that expression of a murine retrovirus capsid-staphylococcal nuclease fusion protein inhibits multiplication of the corresponding murine leukemia virus by 30- to 100-fold. Staphylococcal nuclease is apparently inactive intracellularly and hence nontoxic to the host cell, but it is active extracellularly because of its requirement for high concentrations of Ca2+ ions. Virions assembled in and shed from cells expressing the fusion protein contain very small amounts of intact viral RNA, as would be predicted for nuclease-mediated inhibition of viral multiplication.

SPT10 and SPT21 are required for transcription of particular histone genes in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae genome contains four loci that encode histone proteins. Two of these loci, HTA1-HTB1 and HTA2-HTB2, each encode histones H2A and H2B. The other two loci, HHT1-HHF1 and HHT2-HHF2, each encode histones H3 and H4. Because of their redundancy, deletion of any one histone locus does not cause lethality. Previous experiments demonstrated that mutations at one histone locus, HTA1-HTB1, do cause lethality when in conjunction with mutations in the SPT10 gene. SPT10 has been shown to be required for normal levels of transcription of several genes in S. cerevisiae. Motivated by this double-mutant lethality, we have now investigated the interactions of mutations in SPT10 and in a functionally related gene, SPT21, with mutations at each of the four histone loci. These experiments have demonstrated that both SPT10 and SPT21 are required for transcription at two particular histone loci, HTA2-HTB2 and HHF2-HHT2, but not at the other two histone loci. These results suggest that under some conditions, S. cerevisiae may control the level of histone proteins by differential expression of its histone genes.

The SPT10 and SPT21 genes of Saccharomyces cerevisiae.

Mutations in the SPT10 and SPT21 genes were originally isolated as suppressors of Ty and LTR (delta) insertion mutations in Saccharomyces cerevisiae, and the genes were shown to be required for normal transcription at a number of loci in yeast. Now we have cloned, sequenced, mapped and mutagenized SPT10 and SPT21. Since the spt10 mutation used to clone SPT10 resulted in very poor transformation efficiency, a novel method making use of the kar1-1 mutation was used. Neither SPT gene is essential for growth, and constructed null alleles cause phenotypes similar to those caused by spontaneous mutations in the genes. spt10 null alleles are strong suppressor mutations and cause extremely slow growth. Certain spt10 spontaneous alleles are good suppressors but have a normal growth rate, suggesting that the SPT10 protein may have two distinct functions. An amino acid sequence motif that is similar to the Zn-finger motif was found in SPT10. Mutation of the second Cys residue in this motif resulted in loss of complementation of the suppression phenotype but a normal growth rate. Thus, this motif may reside in a part of the SPT10 protein that is important for transcriptional regulation but not for normal growth. Both the SPT10 and SPT21 proteins are relatively tolerant of large deletions; in both cases deletions of the C-terminus resulted in at least partially functional proteins; also, a large internal deletion in SPT21 was phenotypically wild type.

Hotspots for unselected Ty1 transposition events on yeast chromosome III are near tRNA genes and LTR sequences.

A collection of yeast strains bearing single marked Ty1 insertions on chromosome III was generated. Over 100 such insertions were physically mapped by pulsed-field gel electrophoresis. These insertions are very nonrandomly distributed. Thirty-two such insertions were cloned by the inverted PCR technique, and the flanking DNA sequences were determined. The sequenced insertions all fell within a few very limited regions of chromosome III. Most of these regions contained tRNA coding regions and/or LTRs of preexisting transposable elements. Open reading frames were disrupted at a far lower frequency than expected for random transposition. The results suggest that the Ty1 integration machinery can detect regions of the genome that may represent "safe havens" for insertion. These regions of the genome do not contain any special DNA sequences, nor do they behave as particularly good targets for Ty1 integration in vitro, suggesting that the targeted regions have special properties allowing specific recognition in vivo.

The products of the SPT10 and SPT21 genes of Saccharomyces cerevisiae increase the amplitude of transcriptional regulation at a large number of unlinked loci.

The 3' long terminal repeat (LTR) of yeast transposon Ty1 is not normally used as a promoter, although it contains sequences identical to those found in the 5' LTR, which does act as a promoter. We have isolated mutations that fall into two genes, SPT10 and SPT21, that allow the 3' LTRs of Ty1 elements inserted at various positions in the genome of Saccharomyces cerevisiae to act as promoters. We find that mutations in these two genes alter transcriptional regulation of Ty1 LTRs and also of certain non-Ty1-related promoters in two ways: (i) they allow the low-level expression of several genes under repressing conditions, and (ii) they allow transcription from the 5' LTR of Ty1 elements in the absence of a normally required activator, SPT3. Furthermore, the fully induced levels of transcription of several genes are reduced in these spt mutants. Hence, the products of these two genes increase the amplitude of transcriptional regulation of a wide variety of unlinked loci.

Doubling Ty1 element copy number in Saccharomyces cerevisiae: host genome stability and phenotypic effects.

Haploid yeast strains bearing approximately double the normal number of Ty1 elements have been constructed using marked GAL/Ty1 fusion plasmids. The strains maintain their high transposon copy number and overall genome structure in the absence of selection. The strains bearing extra Ty1 copies are surprisingly similar phenotypically to the parental strain. The results suggest that the limit to transposon copy number, if any, has not been reached. When these strains are crossed by wild-type strains (i.e., bearing the normal complement of Ty1 elements) or by strains of opposite mating type also bearing excess Ty1 elements, normal to very slightly reduced spore viability is observed, indicating that increasing the extent of transposon homology scattered around the genome does not result in significant increases in frequency of ectopic reciprocal recombination. The results suggest that yeast cells have evolved mechanisms for coping with excess transposon copies in the genome.

New antiviral strategy using capsid-nuclease fusion proteins.

Overexpression of dominant-negative mutants of various viral proteins can result in 'intracellular immunization'. Here we describe a new approach to interfering with viral replication in which a nuclease is fused to a capsid component so that the nuclease is encapsidated inside the virion where it can inactivate viral nucleic acid. We used Ty1, a yeast retrotransposon whose transposition closely parallels retroviral replication mechanisms and serves as an easily manipulated model for the retroviral infection process. We constructed fusion genes consisting of the region encoding the N-terminal portion of the TYA/TYB open reading frames of retrotransposon Ty1 and either of two different nuclease genes. Ty1-nuclease fusion proteins are targeted to Ty1 virus-like particles, and are active in degrading nucleic acids. A Ty1-barnase fusion protein causes 98-99% reduction in the efficiency of Ty1 transposition in vivo, presumably by degrading encapsidated Ty1 RNA. This strategy, referred to as capsid-targeted viral inactivation, may be useful for interfering with the replication of retroviruses and other viruses.

Ty1 transposition in Saccharomyces cerevisiae is nonrandom.

A large collection of Ty1 insertions in the URA3 and LYS2 loci was generated using a GAL1-Ty1 fusion to augment the transposition frequency. The sites of insertion of most of these Ty elements were sequenced. There appears to be a gradient of frequency of insertion from the 5' end (highest frequency) to the 3' end (lowest frequency) of both loci. In addition we observed hotspots for transposition. Twelve of the 82 Ty1 insertions in the URA3 locus were inserted in exactly the same site. Hotspots were also observed in the LYS2 locus. All hotspots were in the transcribed part of the genes. Alignment of the sites of insertion and of the neighboring sequences only reveals very weak sequence similarities.

5-Fluoroorotic acid as a selective agent in yeast molecular genetics.

5-FOA is an extremely useful reagent for the selection of Ura- cells amid a population of Ura+ cells. The selection is effective in transformation and recombination studies where loss of URA3+ is desired. A new plasmid shuffling procedure based on the 5-FOAR selection permits the recovery of conditional lethal mutations in cloned genes that encode vital functions.

The HTS1 gene encodes both the cytoplasmic and mitochondrial histidine tRNA synthetases of S. cerevisiae.

The gene encoding the histidine-tRNA synthetase (HTS1) has two in-frame translation start sites located 60 bp apart. One set of HTS1 transcripts (long) initiates upstream of both ATG codons, and the other set (short) initiates between the two ATG codons and therefore contains only the downstream ATG. A mutation that destroys the first AUG on the long message results in the Pet- (respiratory deficient) phenotype, but does not affect either the level of the cytoplasmic histidine-tRNA synthetase or viability. Mutations distal to the second ATG lead to loss of cytoplasmic synthetase function, lethality and respiratory deficiency. These phenotypes can be explained if the longer message were to encode the mitochondrial synthetase and the shorter message were to encode the cytoplasmic histidine-tRNA synthetase.