Virion encapsidation of tRNA(3Lys)-ribozyme chimeric RNAs inhibits HIV infection.

Retroviruses require a specific host cellular tRNA primer for initiation of first-strand DNA synthesis. This primer is bound by viral proteins and copackaged into virions. We have exploited this property in the design and testing of an antiviral ribozyme fused to tRNA(3Lys), the primer used for lentiviral replication, including human immunodeficiency virus (HIV-1 and HIV-2). The chimera consists of tRNA(3Lys) covalently attached to a hammerhead ribozyme, which is targeted to the region immediately upstream of the primer binding site of the HIV-1 genome. The tRNA-ribozyme chimeric transcript is catalytically active in vitro and is efficiently bound by HIV reverse transcriptase with an affinity similar to that of tRNA(3Lys). We have expressed the chimeric RNAs from either the tRNA(3Lys) intragenic RNA polymerase III promoter or from a human U6 snRNA promoter. The U6 promoter results in up to 10-fold enhanced expression of the tRNA-ribozyme. Most importantly, the tRNA(3Lys)-ribozymes are encapsidated in HIV-1 virions such that they are effective in substantially reducing the level of infectious virus produced from cells cotransfected with HIV-1 proviral DNA. These results demonstrate the feasibility of using this novel strategy to reduce HIV infectivity and more generally indicate the potential power of using the retroviral primer tRNAs as tools for expressing and delivering ribozymes and other antiretroviral RNAs to the virion capsid.

A chimeric tRNA(Lys3)-ribozyme inhibits HIV replication following virion assembly.

Co-localization of ribozymes with their appropriate target is one method utilized to increase their effectiveness in vivo. Effective antiviral ribozymes will likely rely on mechanisms which direct the ribozyme to the genomic or subgenomic RNAs. Exploiting the fact that a specific host cellular tRNA primer is bound by viral proteins and co-packaged with viral genomes in newly synthesized virions, ribozymes were fused to the 3'-terminus of tRNA(Lys3) in an attempt to direct their activity to cleave the HIV-1 genome. This chimeric ribozyme is catalytically active in vitro, and is efficiently recognized and bound by HIV-1 reverse transcriptase with affinities similar to tRNA(Lys3). The intragenic RNA polymerase III promoter entity of the tRNA allows for high levels of expression of the tRNA-RBZ and the preferential localization of transcript within the cytoplasm in transfected cells. This ribozyme was effective in reducing the infectivity of a viral stock which was produced from transiently transfected cells bearing the chimeric gene. These results demonstrate the feasibility of using tRNAs as a means of co-localizing ribozymes with their viral genomic RNA targets. The possibility exists to fuse stable RNAs to ribozymes as a means of increasing their stability and localizing them to their appropriate target sites.

Comparison of deoxyoligonucleotide and tRNA(Lys-3) as primers in an endogenous human immunodeficiency virus-1 in vitro reverse transcription/template-switching reaction.

We developed an endogenous in vitro reverse transcription assay to study the properties of priming and template switching during human immunodeficiency virus (HIV) replication. Reactions were primed with HIV reverse transcriptase (RT) and either a deoxyoligonucleotide primer (dPR) or tRNA(Lys-3), the natural primer for reverse transcription. The RNA templates utilized were the actual HIV sequences involved in the first template switch, namely a primer binding sequence (PBS)/U5/R RNA donor template and a R/U3 RNA acceptor template. Reverse transcription reactions using the latter templates and dPR or tRNA(Lys-3) as primers yielded four major products: (-)-strong-stop DNA, a partial template-switched DNA, full template-switched DNA, and a pseudo-PBS-primed product. Use of dPR resulted in three times less template switching than was obtained with tRNA(Lys-3). When reactions were primed with either dPR or tRNA(Lys-3), increases in acceptor:donor template ratios resulted in augmented template switching. Increasing the concentration of RT resulted in increased priming from the PBS but had no effect on the efficiency of template switching. Decreasing the extent of R region overlap resulted in a drop in efficiency of template switching. Decreases in the R region on the donor template also caused a drop in initiation of transcription that was primed by tRNA(Lys-3) from the PBS. In contrast, a corresponding reduction of the R region on the acceptor template had no effect on priming. We conclude that a transcriptional complex of tRNA(Lys-3) and RT may be associated not only with the PBS but also with other cis RNA sequences and secondary structures in a manner essential for efficient priming and template switching.

Biochemical characterization of the p51 sub-unit of human immunodeficiency virus reverse transcriptase in homo- and heterodimeric recombinant forms of the enzyme.

The biochemical properties of the p51 subunit of HIV-1 reverse transcriptase (RT) were studied in order to understand its role in the heterodimeric form p66/p51 found in virions. A recombinant form of RT, p51/p51, expressed in yeast, was purified and characterized. The enzyme was affinity labeled using a 5' modified oligonucleotide primer, covalently linked, that was further elongated in the presence of a radioactive dNTP precursor. We found that the p51 subunit was labeled in the p51/p51 form, thus reflecting its activity, while this subunit was catalytically silent in the heterodimer, since only the p66 subunit was labeled in the latter recombinant form. Processivity studies showed long-sized products synthesized by p51/p51, as in the case of the other RT forms. The effect of primer tRNA(Lys) on the p51/p51 activity showed a strong inhibitory effect in the absence of KCl, similar to that observed with the p66/p51 form, while the same p51/p51 enzyme was strongly stimulated by tRNA(Lys), like RT p66/p66, when KCl was present in the incubation mixture.

Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA(Lys).

Human immunodeficiency virus (HIV) reverse transcriptase (RT) uses host tRNA(Lys) partially annealed to the primer binding site (PBS) as primer for the initiation of cDNA synthesis. When assaying cDNA synthesis with a template-primer complex formed by an RNA fragment carrying the PBS site and bovine tRNA(Lys) we noticed that an excess of primer tRNA inhibited strongly the DNA polymerase activity of a recombinant HIV RT (p66-p51 heterodimeric form) produced in transformed yeast cells. The same inhibitory effect was observed with animal DNA polymerase alpha, while avian retrovirus RT was neither affected by tRNA(Lys) nor by its specific primer tRNA(Trp). Although the strongest inhibition was observed with tRNA(Lys), other tRNas like tRNA(Phe) and tRNA(Trp) inhibited also the HIV RT, whereas tRNAs specific for valine, proline and glycine had no effect on enzyme activity. Digestion of tRNA(Lys) with pancreatic RNase abolished the inhibition; on the other hand T1 RNase digestion had no effect on the inhibition suggesting a role of the anticodon region in this effect. The 12- and 14-mers corresponding to the anticodon regions of the three bovine tRNA(Lys) isoacceptors inhibited RT activity, indicating that at least an important part of the inhibitory effect could be ascribed to this tRNA region. A strong stimulation of DNA polymerase activity was observed when the effect of tRNA(Lys) was assayed on a recombinant HIV reverse transcriptase produced in a protease deficient yeast strain, which leads to the production of an active p66 enzyme. The same tRNAs that inhibited strongly the heterodimeric form stimulated the p66 form of HIV reverse transcriptase. The results suggest that although both enzymatic forms are able to interact with tRNA(Lys) the topography, as well as the functional implications of the interaction between the precursor and the mature form of HIV reverse transcriptase with the tRNA(Lys) primer, are different.