Faculty Bibliography

Cleavage of the Gag and Gag-Pol polyprotein precursors is a critical step in proliferation of retroviruses and retroelements. The Ty1 retroelement of Saccharomyces cerevisiae forms virus-like particles (VLPs) made of the Gag protein. Ty1 Gag is not obviously homologous to the Gag proteins of retroviruses. The apparent molecular mass of Gag is reduced from 58 to 54 kDa during particle maturation. Antibodies raised against the C-terminal peptide of Gag react with the 58-kDa polypeptide but not with the 54-kDa one, indicating that Gag is proteolytically processed at the C terminus. A protease cleavage site between positions 401 and 402 of the Gag precursor was defined by carboxy-terminal sequencing of the processed form of Gag. Certain deletion and substitution mutations in the C terminus of the Gag precursor result in particles that are two-thirds the diameter of the wild-type VLPs. While the Ty1 protease is active in these mutants, their transposition rates are decreased 20-fold compared with that of wild-type Ty1. Thus, the Gag C-terminal portion, released in the course of particle maturation, probably plays a significant role in VLP morphogenesis and Ty1 transposition.

Retroviruses undergo a high frequency of genetic alterations during the process of copying their RNA genomes. However, little is known about the replication fidelity of other elements that transpose via reverse transcription of an RNA intermediate. The complete sequence of 29 independently integrated copies of the yeast retrotransposon Ty1 (173,043 nt) was determined, and the mutation rate during a single cycle of replication was calculated. The observed base substitution rate of 2.5 x 10(-5) bp per replication cycle suggests that this intracellular element can mutate as rapidly as retroviruses. The pattern and distribution of errors in the Ty1 genome is nonrandom and provides clues to potential in vivo molecular mechanisms of reverse transcriptase-mediated error generation, including heterogeneous RNase H cleavage of Ty1 RNA, addition of terminal nontemplated bases, and transient dislocation and realignment of primer-templates. Overall, analysis of errors generated during Ty1 replication underscores the utility of a genetically tractable model system for the study of reverse transcriptase fidelity.

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.

Clinically important mutant p53 proteins may be tumorigenic through a dominant-negative mechanism or due to a gain-of-function. Examples for both hypotheses have been described; however, it remains unclear to what extent they apply to TP53 mutations in general. Here it is shown that the mutational spectrum of dominant-negative p53 mutants selected in a novel yeast assay correlates tightly with p53 mutations in cancer. Two classes of dominant-negative mutations are described; the more dominant one affects codons that are essential for the stabilization of the DNA-binding surface of the p53 core domain and for the direct interaction of p53 with its DNA binding sites. These results predict that the vast majority of TP53 mutations leading to cancer do so in a dominant-negative fashion.

Retroviruses and their relatives, the LTR-containing retrotransposons, integrate newly replicated cDNA copies of their genomes into the genomes of their hosts using element-encoded integrases. Although target site selection is not well understood for this general class of elements, it is becoming clear that some elements target their integration events to very specific regions of their host genomes. Evidence is accumulating that the yeast retrotransposon Ty1 behaves in this manner. Ty1 is found frequently adjacent to tRNA genes in the yeast genome and experimental evidence implicates these regions as preferred integration sites. To determine the basis for Ty1 targeting, we developed an in vivo integration assay using a Ty1 donor plasmid and a second target plasmid that could be used to measure the relative frequency of Ty1 integration into sequences cloned from various regions of the yeast genome. Targets containing genes transcribed by RNA polymerase III (Pol III) were up to several hundredfold more active as integration targets than "cold" sequences lacking such genes. High-frequency targeting was dependent on Pol III transcription, and integration was "region specific," occurring exclusively upstream of the transcription start sites of these genes. Thus, Ty1 has evolved a powerful targeting mechanism, requiring Pol III transcription to integrate its DNA at very specific locations within the yeast genome.

Genomic silencing is a fundamental mechanism of transcriptional regulation, yet little is known about conserved mechanisms of silencing. We report here the discovery of four Saccharomyces cerevisiae homologs of the SIR2 silencing gene (HSTs), as well as conservation of this gene family from bacteria to mammals. At least three HST genes can function in silencing; HST1 overexpression restores transcriptional silencing to a sir2 mutant and hst3 hst4 double mutants are defective in telomeric silencing. In addition, HST3 and HST4 together contribute to proper cell cycle progression, radiation resistance, and genomic stability, establishing new connections between silencing and these fundamental cellular processes.

Reverse transcription in the yeast retrotransposon Ty1 follows the general "rules" of retroviral replication overall. However, some details of the retroviral and Ty1 reverse transcription processes are different. We have identified and determined the structure of plus-strand strong-stop DNA and examined the effect of polypurine tract deletion mutations on its synthesis. Furthermore, we have defined the stop signal for plus-strand strong-stop DNA synthesis as an unusual 2'-O-ribosylated nucleotide in the primer tRNA. Full-length plus-strand strong-stop DNA, following strand transfer, would have a terminal 2-base mismatch with minus-strand DNA. These findings indicate that the mechanism of plus-strand strong-stop DNA transfer in Ty1 differs from that of the retroviral transfer and suggest that full-length plus-strand strong-stop DNA is not a direct intermediate in Ty1 retrotransposition.

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.