Adaptation of the Romanomermis culicivorax CCA-Adding Enzyme to Miniaturized Armless tRNA Substrates

The mitochondrial genome of the nematode Romanomermis culicivorax encodes for miniaturized hairpin-like tRNA molecules that lack D- as well as T-arms, strongly deviating from the consensus cloverleaf. The single tRNA nucleotidyltransferase of this organism is fully active on armless tRNAs, while the human counterpart is not able to add a complete CCA-end. Transplanting single regions of the Romanomermis enzyme into the human counterpart, we identified a beta-turn element of the catalytic core that—when inserted into the human enzyme—confers full CCA-adding activity on armless tRNAs. This region, originally identified to position the 3′-end of the tRNA primer in the catalytic core, dramatically increases the enzyme’s substrate affinity. While conventional tRNA substrates bind to the enzyme by interactions with the T-arm, this is not possible in the case of armless tRNAs, and the strong contribution of the beta-turn compensates for an otherwise too weak interaction required for the addition of a complete CCA-terminus. This compensation demonstrates the remarkable evolutionary plasticity of the catalytic core elements of this enzyme to adapt to unconventional tRNA substrates.

Endogenous Retroviruses Walk a Fine Line between Priming and Silencing

Endogenous retroviruses (ERVs) in mammals are closely related to infectious retroviruses and utilize host tRNAs as a primer for reverse transcription and replication, a hallmark of long terminal repeat (LTR) retroelements. Their dependency on tRNA makes these elements vulnerable to targeting by small RNAs derived from the 3′-end of mature tRNAs (3′-tRFs), which are highly expressed during epigenetic reprogramming and potentially protect many tissues in eukaryotes. Here, we review some key functions of ERV reprogramming during mouse and human development and discuss how small RNA-mediated silencing maintains genome stability when ERVs are temporarily released from heterochromatin repression. In particular, we take a closer look at the tRNA primer binding sites (PBS) of two highly active ERV families in mice and their sequence variation that is shaped by the conflict of successful tRNA priming for replication versus evasion of silencing by 3′-tRFs.

Adaptation of the Romanomermis culicivorax CCA-adding enzyme to miniaturized armless tRNA substrates

The mitochondrial genome of the nematode Romanomermis culicivorax encodes for miniaturized hairpin-like tRNA molecules that lack D- as well as T-arms, strongly deviating from the consensus cloverleaf. The single tRNA nucleotidyltransferase of this organism is fully active on armless tRNAs, while the human counterpart is not able to add a complete CCA-end. Transplanting single regions of the Romanomermis enzyme into the human counterpart, we identified a beta-turn element of the catalytic core that – when inserted into the human enzyme - confers full CCA-adding activity on armless tRNAs. This region, originally identified to position the 3’-end of the tRNA primer in the catalytic core, dramatically increases the enzyme’s substrate affinity. While conventional tRNA substrates bind to the enzyme by interactions with the T-arm, this is not possible in the case of armless tRNAs, and the strong contribution of the beta-turn compensates for an otherwise too weak interaction required for the addition of a complete CCA-terminus. This compensation demonstrates the remarkable evolutionary plasticity of the catalytic core elements of this enzyme to adapt to unconventional tRNA substrates.

Mutations in the U5 Region Adjacent to the Primer Binding Site Affect tRNA Cleavage by Human Immunodeficiency Virus Type 1 Reverse Transcriptase In Vivo

ABSTRACT In retroviruses, the first nucleotide added to the tRNA primer defines the end of the U5 region in the right long terminal repeat, and the subsequent removal of this tRNA primer by RNase H exactly defines the U5 end of the linear double-stranded DNA. In most retroviruses, the entire tRNA is removed by RNase H cleavage at the RNA/DNA junction. However, the RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase cleaves the tRNA 1 nucleotide from the RNA/DNA junction at the U5/primer binding site (PBS) junction, which leaves an rA residue at the U5 terminus. We made sequence changes at the end of the U5 region adjacent to the PBS in HIV-1 to determine whether such changes affect the specificity of tRNA primer cleavage by RNase H. In some of the mutants, RNase H usually removed the entire tRNA, showing that the cleavage specificity was shifted by 1 nucleotide. This result suggests that the tRNA cleavage specificity of the HIV-1 RNase domain H depends on sequences in U5.

tRNA Isoacceptor Preference prior to Retrovirus Gag-Pol Junction Links Primer Selection and Viral Translation

ABSTRACT An essential step in the replication of all retroviruses is the capture of a cellular tRNA that is used as the primer for reverse transcription. The 3′-terminal 18 nucleotides of the tRNA are complementary to the primer binding site (PBS). Moloney murine leukemia virus (MuLV) preferentially captures tRNAPro. To investigate the specificity of primer selection, the PBS of MuLV was altered to be complementary to different tRNAs. Analysis of the infectivity of the virus and stability of the PBS following in vitro replication revealed that MuLV prefers to select tRNAPro, tRNAGly, or tRNAArg. Previous studies from our laboratory have suggested that tRNA primer capture is coordinated with translation. Coincidentally, a cluster of proline, arginine, and glycine precedes the Gag-Pol junction of MuLV. Human immunodeficiency virus type 1 (HIV-1), which prefers \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{3}^{\mathrm{Lys}}\) \end{document} as the primer, can be forced to utilize tRNAMet, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{1,2}^{\mathrm{Lys}}\) \end{document} , tRNAHis, or tRNAGlu, although these viruses replicate poorly. Codons for methionine, lysine, histidine, or glutamic acid are found prior to the Gag-Pol frameshift site. HIV-1 was mutated so that the 5 lysine codons prior to the Gag-Pol frameshift region were specific for \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{1,2}^{\mathrm{Lys}}\) \end{document} . HIV-1 forced to use \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{1,2}^{\mathrm{Lys}}\) \end{document} as the primer, with the mutation of codons specific for \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{1,2}^{\mathrm{Lys}}\) \end{document} prior to the Gag-Pol junction, had enhanced infectivity and replicated similarly to the wild-type virus. The results demonstrate that codon preference prior to the Gag-Pol junction influences primer selection and suggest a coordination of Gag-Pol synthesis and acquisition of the tRNA primer required for retrovirus replication.

Identification and Characterization of an Exogenous Retrovirus from Atlantic Salmon Swim Bladder Sarcomas

ABSTRACT A novel piscine retrovirus has been identified in association with an outbreak of leiomyosarcoma in the swim bladders of Atlantic salmon. The complete nucleotide sequence of the Atlantic salmon swim bladder sarcoma virus (SSSV) provirus is 10.9 kb in length and shares a structure and transcriptional profile similar to those of murine leukemia virus-like simple retroviruses. SSSV appears unique to simple retroviruses by not harboring sequences in the Atlantic salmon genome. Additionally, SSSV differs from other retroviruses in potentially utilizing a methionine tRNA primer binding site. SSSV-associated tumors contain high proviral copy numbers (greater than 30 per cell) and a polyclonal integration pattern. Phylogenetic analysis based on reverse transcriptase places SSSV with zebrafish endogenous retrovirus (ZFERV) between the Gammaretrovirus and Epsilonretrovirus genera. Large regions of continuous homology between SSSV and ZFERV Gag, Pol, and Env suggest that these viruses represent a new group of related piscine retroviruses.