Pol Genes Of Retroviruses Research Articles

Cis-Allosteric Regulation of HIV-1 Reverse Transcriptase by Integrase.

Reverse transcriptase (RT) and integrase (IN) are encoded tandemly in the pol genes of retroviruses. We reported recently that HIV-1 RT and IN need to be supplied as the pol precursor intermediates, in which RT and IN are in fusion form (RTIN) to exert efficient reverse transcription in the context of HIV-1 replication. The mechanism underlying RTIN's effect, however, remains to be elucidated. In this study, we examined the effect of IN fusion on RT during reverse transcription by an in vitro cell-free assay, using recombinant HIV-1 RTIN (rRTIN). We found that, compared to recombinant RT (rRT), rRTIN generated significantly higher cDNAs under physiological concentrations of dNTPs (less than 10 μM), suggesting increased affinity of RTIN to dNTPs. Importantly, the cleavage of RTIN with HIV-1 protease reduced cDNA levels at a low dose of dNTPs. Similarly, sensitivities against RT inhibitors were significantly altered in RTIN form. Finally, analysis of molecular dynamics simulations of RT and RTIN suggested that IN can influence the structural dynamics of the RT active center and the inhibitor binding pockets in cis. Thus, we demonstrated, for the first time, the cis-allosteric regulatory roles of IN in RT structure and enzymatic activity.

Identification and analysis of the gag-pol ribosomal frameshift site of feline immunodeficiency virus

The pol genes of retroviruses are translated as gag-pol fusion proteins by ribosomal frameshifting within the gag-pol overlap region. During the ribosomal frameshift event, the gag open reading frame is shifted -1 nt to allow in-phase reading of the pol open reading frame. A consensus frameshift signal sequence of GGGAAAC within the gag-pol overlap region of feline immunodeficiency virus (FIV) has been identified followed by a sequence that has the potential for a pseudoknot tertiary structure. Using recombinant baculoviruses in which the frameshift occurs efficiently, the consensus sequence has been shown to be the site of the frameshift event. A mutation creating a termination codon just downstream of the putative frameshift signal sequence but upstream of the potential pseudoknot structure made a shorter gag product, but did not affect the efficiency of frameshifting. A mutation creating a termination codon just upstream of the putative frameshift signal made a shorter product and essentially abrogated frameshifting. Mutations in the first stem or the second stem in the potential pseudoknot structure severely reduced the frameshifting efficiency. Mutations which altered the length between the frameshift signal and the pseudoknot structure (the so-called spacer region) also reduced the frameshift efficiency. The insertion of a palindromic sequence, which could form a hairpin structure just upstream of the frameshift signal sequence, also affected the frameshifting. These results support the view that the ribosomal frameshift event in the FIV gag-pol region involves the identified signal sequence and appears to require the precisely positioned downstream sequence and indicated pseudoknot structure for efficient frameshifting.

A new member of a family of site-specific retrotransposons is present in the spliced leader RNA genes of Trypanosoma cruzi.

A new member of a family of site-specific retrotransposons is described in the New World trypanosome Trypanosoma cruzi. This element, CZAR (cruzi-associated retrotransposon), resembles two previously described retrotransposons found in the African trypanosome T. brucei gambiense and the mosquito trypanosomatid Crithidia fasciculata in specifically inserting between nucleotides 11 and 12 of the highly conserved 39-mer of the spliced leader RNA (SL-RNA) gene. CZAR is similar in overall organization to the other two SL-RNA-associated elements. It possesses two potential long open reading frames which resemble the gag and pol genes of retroviruses. In the pol open reading frame, all three elements contain similarly arranged endonuclease domains and share extensive amino acid homology in the reverse transcriptase region. All are associated with the SL-RNA gene locus and are present in low copy numbers. They do not appear to have 5' truncated versions. All three retrotransposons are otherwise quite distinct from one another, with no significant overall amino acid homology. The presence of such retroelements inserted into the identical site within SL-RNA gene sequences in at least three evolutionarily distant trypanosomatid species argues for a functional role. Because these elements appear to have a precise target site requirement for integration, we refer to them as SL siteposons.

A New Member of a Family of Site-Specific Retrotransposons Is Present in the Spliced Leader RNA Genes of Trypanosoma cruzi

A new member of a family of site-specific retrotransposons is described in the New World trypanosome Trypanosoma cruzi. This element, CZAR (cruzi-associated retrotransposon), resembles two previously described retrotransposons found in the African trypanosome T. brucei gambiense and the mosquito trypanosomatid Crithidia fasciculata in specifically inserting between nucleotides 11 and 12 of the highly conserved 39-mer of the spliced leader RNA (SL-RNA) gene. CZAR is similar in overall organization to the other two SL-RNA-associated elements. It possesses two potential long open reading frames which resemble the gag and pol genes of retroviruses. In the pol open reading frame, all three elements contain similarly arranged endonuclease domains and share extensive amino acid homology in the reverse transcriptase region. All are associated with the SL-RNA gene locus and are present in low copy numbers. They do not appear to have 5' truncated versions. All three retrotransposons are otherwise quite distinct from one another, with no significant overall amino acid homology. The presence of such retroelements inserted into the identical site within SL-RNA gene sequences in at least three evolutionarily distant trypanosomatid species argues for a functional role. Because these elements appear to have a precise target site requirement for integration, we refer to them as SL siteposons.

The site-specific ribosomal insertion element type II of Bombyx mori (R2Bm) contains the coding sequence for a reverse transcriptase-like enzyme.

Two classes of DNA elements interrupt a fraction of the rRNA repeats of Bombyx mori. We have analyzed by genomic blotting and sequence analysis one class of these elements which we have named R2. These elements occupy approximately 9% of the rDNA units of B. mori and appear to be homologous to the type II rDNA insertions detected in Drosophila melanogaster. Approximately 25 copies of R2 exist within the B. mori genome, of which at least 20 are located at a precise location within otherwise typical rDNA units. Nucleotide sequence analysis has revealed that the 4.2-kilobase-pair R2 element has a single large open reading frame, occupying over 82% of the total length of the element. The central region of this 1,151-amino-acid open reading frame shows homology to the reverse transcriptase enzymes found in retroviruses and certain transposable elements. Amino acid homology of this region is highest to the mobile line 1 elements of mammals, followed by the mitochondrial type II introns of fungi, and the pol gene of retroviruses. Less homology exists with transposable elements of D. melanogaster and Saccharomyces cerevisiae. Two additional regions of sequence homology between L1 and R2 elements were also found outside the reverse transcriptase region. We suggest that the R2 elements are retrotransposons that are site specific in their insertion into the genome. Such mobility would enable these elements to occupy a small fraction of the rDNA units of B. mori despite their continual elimination from the rDNA locus by sequence turnover.

The Site-Specific Ribosomal Insertion Element Type II of Bombyx mori (R2Bm) Contains the Coding Sequence for a Reverse Transcriptase-Like Enzyme

Two classes of DNA elements interrupt a fraction of the rRNA repeats of Bombyx mori. We have analyzed by genomic blotting and sequence analysis one class of these elements which we have named R2. These elements occupy approximately 9% of the rDNA units of B. mori and appear to be homologous to the type II rDNA insertions detected in Drosophila melanogaster. Approximately 25 copies of R2 exist within the B. mori genome, of which at least 20 are located at a precise location within otherwise typical rDNA units. Nucleotide sequence analysis has revealed that the 4.2-kilobase-pair R2 element has a single large open reading frame, occupying over 82% of the total length of the element. The central region of this 1,151-amino-acid open reading frame shows homology to the reverse transcriptase enzymes found in retroviruses and certain transposable elements. Amino acid homology of this region is highest to the mobile line 1 elements of mammals, followed by the mitochondrial type II introns of fungi, and the pol gene of retroviruses. Less homology exists with transposable elements of D. melanogaster and Saccharomyces cerevisiae. Two additional regions of sequence homology between L1 and R2 elements were also found outside the reverse transcriptase region. We suggest that the R2 elements are retrotransposons that are site specific in their insertion into the genome. Such mobility would enable these elements to occupy a small fraction of the rDNA units of B. mori despite their continual elimination from the rDNA locus by sequence turnover.