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A number of abundant mobile genetic elements called retrotransposons reverse transcribe RNA to generate DNA for insertion into eukaryotic genomes. Four major classes of retrotransposons are described here. First, the long-terminal-repeat (LTR) retrotransposons have similar structures and mechanisms to those of the vertebrate retroviruses. Genes that may enable these retrotransposons to leave a cell have been acquired by these elements in a number of animal and plant lineages. Second, the tyrosine recombinase retrotransposons are similar to the LTR retrotransposons except that they have substituted a recombinase for the integrase and recombine into the host chromosomes. Third, the non-LTR retrotransposons use a cleaved chromosomal target site generated by an encoded endonuclease to prime reverse transcription. Finally, the Penelope-like retrotransposons are not well understood but appear to also use cleaved DNA or the ends of chromosomes as primer for reverse transcription. Described in the second part of this review are the enzymatic properties of the reverse transcriptases (RTs) encoded by retrotransposons. The RTs of the LTR retrotransposons are highly divergent in sequence but have similar enzymatic activities to those of retroviruses. The RTs of the non-LTR retrotransposons have several unique properties reflecting their adaptation to a different mechanism of retrotransposition.

We compared the mechanistic and kinetic properties of murine leukemia virus (MuLV) and human immunodeficiency virus type 1 (HIV-1) reverse transcriptases (RTs) during RNA-dependent DNA polymerization and mutation synthesis using pre-steady-state kinetic analysis. First, MuLV RT showed 6.5-121.6-fold lower binding affinity (K d ) to deoxynucleotide triphosphate (dNTP) substrates than HIV-1 RT, although the two RTs have similar incorporation rates (k pol ). Second, compared with HIV-1 RT, MuLV RT showed dramatic reduction during multiple dNTP incorporations at low dNTP concentrations. Presumably, due to its low dNTP binding affinity, the dNTP binding step becomes rate-limiting in the multiple rounds of the dNTP incorporation by MuLV RT, especially at low dNTP concentrations. Third, similar fold differences between MuLV and HIV-1 RTs in the K d and k pol values to correct and incorrect dNTPs were observed. This indicates that these two RT proteins have similar misinsertion fidelities. Fourth, these two RT proteins have different mechanistic capabilities regarding mismatch extension. MuLV RT has a 3.1-fold lower mismatch extension fidelity, compared with HIV-1 RT. Finally, MuLV RT has a 3.8-fold lower binding affinity to mismatched template/primer (T/P) substrate compared with HIV-1 RT. Our data suggest that the active site of MuLV RT has an intrinsically low dNTP binding affinity, compared with HIV-1 RT. In addition, instead of the misinsertion step, the mismatch extension step, which varies between MuLV and HIV-1 RTs, contributes to their fidelity differences. The implications of these kinetic differences between MuLV and HIV-1 RTs on viral cell type specificity and mutagenesis are discussed.Retroviruses encode a versatile DNA polymerase called reverse transcriptase (RT).1 The function of RT is to synthesize linear double-stranded proviral DNAs from single-stranded positive sense viral RNA genomes during viral replication. In order to catalyze this process, RTs perform several distinct enzymatic reactions including RNA-dependent DNA polymerization, DNA-dependent DNA polymerization, strand transfer, and RNase H cleavage (1). The DNA polymerase activity of RTs has been targeted, using various types of RT inhibitors such as nucleoside substrate-like compounds (i.e. azidothymidine (AZT) and didanosine (ddI)) (2), as a means to reduce viral replication in infected individuals. Lentiviruses such as human immunodeficiency virus type 1 (HIV-1) uniquely infect terminally differentiated/nondividing cells (i.e. macrophages) as well as dividing cells (i.e. activated CD4ϩ T cells), whereas oncoretroviruses such as murine leukemia virus (MuLV) productively replicate mainly in dividing cells (3,4). Numerous studies have reported that actively dividing cells have higher cellular deoxynucleotide triphosphate (dNTP) concentrations than nondividing cells (5). Recently, it was reported that the cellular dNTP concentration of human macrophages (ϳ40 nM) is ϳ100 times lower than that of dividing CD4 ϩ T cells (ϳ5 M) (6). Therefore, RTs ...

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R2 elements are non-long terminal repeat (non-LTR) retrotransposons with a single open reading-frame encoding reverse transcriptase, DNA endonuclease and nucleic acid-binding domains. The elements are specialized for insertion into the 28 S rRNA genes of many animal phyla. The R2-encoded activities initiate retrotransposition by sequence-specific cleavage of the 28 S gene target site and the utilization of the released DNA 3' end to prime reverse transcription (target primed reverse transcription). The activity of the R2 polymerase on RNA templates has been shown to differ from retroviral reverse transcriptases (RTs) in a number of properties. We demonstrate that the R2-RT is capable of efficiently utilizing single-stranded DNA (ssDNA) as a template. The processivity of the enzyme on ssDNA templates is higher than its processivity on RNA templates. This finding suggests that R2-RT is also capable of synthesizing the second DNA strand during retrotransposition. However, R2-RT lacks the RNAse H activity that is typically used by retroviral and LTR-retrotransposon RTs to remove the RNA strand before the first DNA strand is used as template. Remarkably, R2-RT can displace RNA strands that are annealed to ssDNA templates with essentially no loss of processivity. Such strand displacement activity is highly unusual for a DNA polymerase. Thus the single R2 protein contains all the activities needed to make a double-stranded DNA product from an RNA transcript. Finally, during these studies we found an unexpected property of the highly sequence-specific R2 endonuclease domain. The endonuclease can non-specifically cleave ssDNA at a junction with double-stranded DNA. This activity suggests that second-strand cleavage of the target site may not be sequence specific, but rather is specified by a single-stranded region generated when the first DNA strand is used to prime reverse transcription.

We characterized HIV-1 reverse transcriptase (RT) variants either with or without the (؊)-2,3-deoxy-3-thiacytidine-resistant M184I mutation isolated from a single HIV-1 infected patient. First, unlike variants with wild-type M184, M184I RT variants displayed significantly reduced DNA polymerase activity at low dNTP concentrations, which is indicative of reduced dNTP binding affinity. Second, the M184I variant displayed a ϳ10-to 13-fold reduction in dNTP binding affinity, compared with the Met-184 variant. However, the k pol values of these two RTs were similar. Third, unlike HIV-1 vectors with wild-type RT, the HIV-1 vector harboring M184I RT failed to transduce cell types containing low dNTP concentrations, such as human macrophage, likely due to the reduced DNA polymerization activity of the M184I RT under low cellular dNTP concentration conditions. Finally, we compared the binary complex structures of wild-type and M184I RTs. The Ile mutation at position 184 with a longer and more rigid ␤-branched side chain, which was previously known to alter the RT-template interaction, also appears to deform the shape of the dNTP binding pocket. This can restrict ground state dNTP binding and lead to inefficient DNA synthesis particularly at low dNTP concentrations, ultimately contributing to viral replication failure in macrophage and instability in vivo of the M184I mutation.(Ϫ)-2Ј,3Ј-Deoxy-3Ј-thiacytidine (3TC), 3 a deoxycytidine analog reverse transcriptase (RT) inhibitor, has been routinely included in the anti-HIV-1 drug regime (1, 2). During 3TC therapy, two amino acid substitutions at position 184 of RT are sequentially selected, conferring viral resistance to this drug. The M184I mutation is detected earlier, and eventually replaced with the M184V mutation. One initial explanation for the delayed selection of the M184V mutation is the requirement of two nucleotide mutations to develop the final M184V mutation from the wild-type methionine codon (ATG). In contrast, the M184I mutation requires only a single nucleotide mutation from the Met codon, which may result in its early selection during 3TC therapy (3-7).Structural analysis later provided a functional explanation for the instability in vivo of the M184I mutation. The M184I mutation alters the RT interaction with the template nucleotide more significantly than the M184V mutation, leading to more severely decreased processivity, compared with the M184V RT (8). Indeed, the distributive DNA synthesis catalyzed by the M184I RT has been a major explanation for the instability in vivo of this mutation and its rapid transition to the M184V mutation. The long ␤-branched side chains of these two mutations efficiently block the entry of 3TCTP to the active site (8, 9). Interestingly, pre-steady-state kinetic studies of the M184V mutant demonstrated that the M184V mutation only slightly (1-to 2-fold) affects K d (dNTP binding affinity), but not k pol (conformational change/chemical catalysis) steps of HIV-1 RT with normal dNTP substrates, implying that the Val mutation d...

R2 non-LTR retrotransposons insert at a specific site in the 28S rRNA genes of many animal phyla. R2 elements encode a single polypeptide with reverse transcriptase, endonuclease and nucleic acid binding domains. Integration involves separate cleavage of the two DNA strands at the target site and utilization of the released 3′ ends to prime DNA synthesis. Critical to this integration is the ability of the protein to specifically bind 3′ and 5′ regions of the R2 RNA. In this report, alanine mutations in two conserved motifs N-terminal to the reverse transcriptase domain were generated and shown to result in proteins that retained the ability to cleave the first strand of the DNA target, to reverse transcribe RNA from an annealed primer and to displace annealed RNA when using DNA as a template. However, the mutant proteins had greatly reduced ability to bind 3′ and 5′ RNA in mobility shift assays, use the DNA target to prime reverse transcription and conduct second-strand DNA cleavage. These motifs thus appear to participate in all activities of the R2 protein known to require specific RNA binding. The similarity of these R2 RNA binding motifs to those of telomerase and group II introns is discussed.

Two previously identified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants, Q151N and V148I, are known to have reduced dNTP binding affinity but possess wild-type chemical catalysis rates. Structural modeling based on the crystal structure of the HIV-1 RT ternary complex with dTTP proposes that Q151N loses the interaction with the 3'-OH of the incoming dTTP and that V148I disrupts positioning of Q151 for this interaction. On the basis of this, we predicted that while wild-type (WT) HIV-1 RT would have decreased binding affinity to dTTP analogues lacking 3'-OH, compared to dTTP, the Q151N and V148I RT mutants should have decreased but similar affinity to both dTTP and dTTP analogues. Pre-steady-state kinetics on WT RT showed 14- and 53-fold higher K(d) values for the 3'-OH lacking ddTTP and acyTTP, compared to dTTP. In contrast, the Q151N and V148I mutants, which were predicted to have lost H-bonding interaction with the 3'-OH of dTTP, showed higher but similar K(d) values for dTTP, ddTTP, and acyTTP. Interestingly, the Q151N and V148I RTs bound to AZTTP approximately 12 and 18 times more tightly than to dTTP, respectively. Our structure modeling suggests that these RT mutants can interact with the azido moiety of AZTTP, which is 1.4 A longer than the 3'-OH of dTTP. The kinetic data presented in this report demonstrate the functional role of the Q151 residue in HIV-1 RT interaction with dTTP and its analogues containing chemical modifications at the 3'-C of the sugar moiety.