tRNA Primer Sequestration as an Antiviral Strategy

2013 ◽  
pp. 205-221
Author(s):  
Christopher P. Jones ◽  
Karin Musier-Forsyth
Keyword(s):  
Trna Primer

Proviral sequences that restrict retroviral expression in mouse embryonal carcinoma cells

1987 ◽  
Vol 7 (10) ◽  
pp. 3775-3784
Author(s):  
T P Loh ◽  
L L Sievert ◽  
R W Scott
Keyword(s):  
Long Terminal Repeat ◽  
Transient Expression ◽  
Embryonal Carcinoma ◽  
Leukemia Virus ◽  
Terminal Repeat ◽  
Moloney Murine Leukemia Virus ◽  
Primer Binding Site ◽  
Promoter Sequences ◽  
Trna Primer ◽  
Ec Cells

Embryonal carcinoma (EC) cells are nonpermissive for retrovirus replication. Restriction of retroviral expression in EC cells was studied by using DNA transfection techniques. To investigate the activity of the Moloney murine leukemia virus (M-MuLV)enhancer and promoter sequences, the M-MuLV long terminal repeat and the defined long terminal repeat deletions were linked to neo structural gene sequences that encode resistance to the neomycin analog G418. Transient expression data and drug resistance frequencies support the findings that the M-MuLV enhancer is not active in EC cells but that promoter sequences are functional. In addition, a proviral DNA fragment that encodes the leader RNA sequence of a M-MuLV recombinant retrovirus was found to restrict expression specifically in EC cells. Deletion analysis of the leader fragment localized the inhibitory sequences to a region that spans the M-MuLV tRNA primer binding site. It is not known whether restriction occurs at a transcriptional or posttranscriptional level, but steady-state RNA levels in transient expression assays were significantly reduced.

scholarly journals Improved transfer of the leukocyte integrin CD18 subunit into hematopoietic cell lines by using retroviral vectors having a gibbon ape leukemia virus envelope

Blood ◽  
1995 ◽  
Vol 86 (6) ◽  
pp. 2379-2387 ◽  
Author(s):  
TR Jr Bauer ◽  
AD Miller ◽  
DD Hickstein
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Leukemia Virus ◽  
K562 Cells ◽  
Retroviral Vectors ◽  
Packaging Cell Line ◽  
Virus Receptor ◽  
Packaging Cell ◽  
Trna Primer ◽  
Gibbon Ape Leukemia Virus

Leukocyte adherence deficiency (LAD) is an inherited immunodeficiency disease caused by defects in the CD18 leukocyte integrin subunit. Transduction of CD18 into hematopoietic cells from children with LAD represents a potential therapy for this disorder. In an attempt to maximize transfer and expression of CD18, we evaluated retroviral vectors with and without the neomycin selectable marker, with a modified tRNA primer binding site designed to prevent inhibition of gene expression, and with two different viral envelope proteins produced by using the amphotropic retrovirus packaging cell line PA317 or the gibbon ape leukemia virus packaging cell line PG13. The vectors were tested using transducing K562/CD11b cells and LAD Epstein-Barr virus (EBV) B cells and measuring levels of cell-surface CD11/CD18 expression by fluorescence-activated cell sorter analysis. The best results were obtained with vectors made using PG13 packaging cells, for which about 25% of the K562 cells exposed once to the vectors expressed surface CD11b/CD18 and about 25% of the LAD EBV B cells exposed three times over a 3-day period to the vectors expressed surface CD11a/CD18. In contrast, transduction of cells under similar conditions with retroviral vectors produced using PA317 producer cells yielded less than 2% of the K562 cells and less than 4% of the LAD EBV B cells expressing the CD11/CD18 heterodimer on the cell surface. The presence or absence of the neomycin resistance gene or the modified tRNA primer had no effect on CD18 gene transfer rate or expression level. The increase in transduction with PG13 vectors correlated with Northern blotting and reverse transcription-polymerase chain reaction studies that indicated that both K562 cells and the LAD EBV B cells express transcripts for the gibbon ape leukemia virus receptor at higher levels than for the amphotropic virus receptor. These findings indicate that the transduction efficiency of retroviral packaging cell lines correlates with receptor gene expression in the target cells and that vectors made using PG13 cells may be efficacious for gene therapy for LAD and other diseases in which gene transfer to hematopoietic cells is required.

scholarly journals Endogenous Retroviruses Walk a Fine Line between Priming and Silencing

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 792 ◽  
Author(s):  
Harrison Cullen ◽  
Andrea J. Schorn
Keyword(s):  
Small Rna ◽  
Binding Sites ◽  
Small Rnas ◽  
Genome Stability ◽  
Sequence Variation ◽  
Endogenous Retroviruses ◽  
Highly Active ◽  
Trna Primer ◽  
Ltr Retroelements ◽  
Transcription And Replication

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.

scholarly journals Selection of Retroviral Reverse Transcription Primer Is Coordinated with tRNA Biogenesis

2003 ◽  
Vol 77 (16) ◽  
pp. 8695-8701 ◽  
Author(s):  
Nathan J. Kelly ◽  
Matthew T. Palmer ◽  
Casey D. Morrow
Keyword(s):  
Reverse Transcription ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Leukemia Virus ◽  
Wild Type ◽  
Primer Selection ◽  
Yeast Trna ◽  
Trna Primer ◽  
Hiv 1 ◽  
Selection Of

ABSTRACT Initiation of retrovirus reverse transcription requires the selection of a tRNA primer from the intracellular milieu. To investigate the features of primer selection, a human immunodeficiency virus type 1 (HIV-1) and a murine leukemia virus (MuLV) were created that require yeast tRNAPhe to be supplied in trans for infectivity. Wild-type yeast tRNAPhe expressed in mammalian cells was transported to the cytoplasm and aminoacylated. In contrast, tRNAPhe without the D loop (tRNAPheD−) was retained within the nucleus and did not complement infectivity of either HIV-1 or MuLV; however, infectivity was restored when tRNAPheD− was directly transfected into the cytoplasm of cells. A tRNAPhe mutant (tRNAPheUUA) that did not have the capacity to be aminoacylated was transported to the cytoplasm and did complement infectivity of both HIV-1 and MuLV, albeit at a level less than that with wild-type tRNAPhe. Collectively, our results demonstrate that the tRNA primer captured by HIV-1 and MuLV occurs after nuclear export of tRNA and supports a model in which primer selection for retroviruses is coordinated with tRNA biogenesis at the intracellular site of protein synthesis.

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

2020 ◽  
Vol 21 (23) ◽  
pp. 9047
Author(s):  
Oliver Hennig ◽  
Susanne Philipp ◽  
Sonja Bonin ◽  
Kévin Rollet ◽  
Tim Kolberg ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Weak Interaction ◽  
Substrate Affinity ◽  
Catalytic Core ◽  
Beta Turn ◽  
Human Enzyme ◽  
Human Counterpart ◽  
Core Elements ◽  
Trna Primer

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.

scholarly journals Forced Selection of a Human Immunodeficiency Virus Type 1 Variant That Uses a Non-Self tRNA Primer for Reverse Transcription: Involvement of Viral RNA Sequences and the Reverse Transcriptase Enzyme

2004 ◽  
Vol 78 (19) ◽  
pp. 10706-10714 ◽  
Author(s):  
Truus E. M. Abbink ◽  
Nancy Beerens ◽  
Ben Berkhout
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Viral Rna ◽  
Replication Capacity ◽  
Immunodeficiency Virus ◽  
Trna Primer

ABSTRACT Human immunodeficiency virus type 1 uses the tRNA3 Lys molecule as a selective primer for reverse transcription. This primer specificity is imposed by sequence complementarity between the tRNA primer and two motifs in the viral RNA genome: the primer-binding site (PBS) and the primer activation signal (PAS). In addition, there may be specific interactions between the tRNA primer and viral proteins, such as the reverse transcriptase (RT) enzyme. We constructed viruses with mutations in the PAS and PBS that were designed to employ the nonself primer tRNAPro or tRNA1,2 Lys. These mutants exhibited a severe replication defect, indicating that additional adaptation of the mutant virus is required to accommodate the new tRNA primer. Multiple independent virus evolution experiments were performed to select for fast-replicating variants. Reversion to the wild-type PBS-lys3 sequence was the most frequent escape route. However, we identified one culture in which the virus gained replication capacity without reversion of the PBS. This revertant virus eventually optimized the PAS motif for interaction with the nonself primer. Interestingly, earlier evolution samples revealed a single amino acid change of an otherwise well-conserved residue in the RNase H domain of the RT enzyme, implicating this domain in selective primer usage. We demonstrate that both the PAS and RT mutations improve the replication capacity of the tRNA1,2 Lys-using virus.

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

2007 ◽  
Vol 82 (2) ◽  
pp. 719-727 ◽  
Author(s):  
Jangsuk Oh ◽  
Mary Jane McWilliams ◽  
John G. Julias ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Binding Site ◽  
Rnase H ◽  
Primer Binding Site ◽  
Cleavage Specificity ◽  
Immunodeficiency Virus ◽  
Trna Primer ◽  
Hiv 1

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.

scholarly journals The tRNA Primer Activation Signal in the Human Immunodeficiency Virus Type 1 Genome Is Important for Initiation and Processive Elongation of Reverse Transcription

2002 ◽  
Vol 76 (5) ◽  
pp. 2329-2339 ◽  
Author(s):  
Nancy Beerens ◽  
Ben Berkhout
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Sequence Motif ◽  
Immunodeficiency Virus ◽  
Trna Primer ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) reverse transcription is primed by the cellular tRNA3 Lys molecule, which binds, with its 3"-terminal 18 nucleotides (nt), to a complementary sequence in the viral genome, the primer-binding site (PBS). Besides PBS-anti-PBS pairing, additional interactions between viral RNA sequences and the tRNA primer are thought to regulate the process of reverse transcription. We previously identified a novel 8-nt sequence motif in the U5 region of the HIV-1 RNA genome that is critical for tRNA3 Lys-mediated initiation of reverse transcription in vitro. This motif activates initiation from the natural tRNA3 Lys primer but is not involved in tRNA placement and was therefore termed primer activation signal (PAS). It was proposed that the PAS interacts with the anti-PAS motif in the TΨC arm of tRNA3 Lys. In this study, we analyzed several PAS-mutated viruses and performed reverse transcription assays with virion-extracted RNA-tRNA complexes. Mutation of the PAS reduced the efficiency of tRNA-primed reverse transcription. In contrast, mutations in the opposing leader sequence that trigger release of the PAS from base pairing stimulated reverse transcription. These results are similar to the reverse transcription effects observed in vitro. We also selected revertant viruses that partially overcome the reverse transcription defect of the PAS deletion mutant. Remarkably, all revertants acquired a single nucleotide substitution that does not restore the PAS sequence but that stimulates elongation of reverse transcription. These combined results indicate that the additional PAS-anti-PAS interaction is needed to assemble an initiation-competent and processive reverse transcription complex.

scholarly journals Replication of Avian Sarcoma Virus In Vivo Requires an Interaction between the Viral RNA and the TψC Loop of the tRNATrp Primer

2002 ◽  
Vol 76 (15) ◽  
pp. 7571-7577 ◽  
Author(s):  
Shannon Morris ◽  
Michael Johnson ◽  
Ed Stavnezer ◽  
Jonathan Leis
Keyword(s):  
Consensus Sequence ◽  
Viral Rna ◽  
Primer Binding Site ◽  
Avian Sarcoma Virus ◽  
Biologically Relevant ◽  
Efficient Replication ◽  
Trna Primer ◽  
Sarcoma Virus ◽  
Avian Sarcoma

ABSTRACT Reverse transcription in avian sarcoma virus (ASV) initiates from the 3′ end of a tRNATrp primer, which anneals near the 5′ end of the RNA genome. The region around the primer-binding site (PBS) forms an elaborate stem structure composed of the U5-inverted repeat (U5-IR) stem, the U5-leader stem, and the association of the tRNA primer with the PBS. There is evidence for an additional interaction between the viral U5 RNA and the TψC loop of the tRNATrp (U5-TψC). We now demonstrate that this U5-TψC interaction is necessary for efficient replication of ASV in culture. By randomizing specific biologically relevant regions of the viral RNA, thereby producing a library of mutant viruses, we are able to select, through multiple rounds of infection, those sequences imparting survival fitness to the virus. Randomizing the U5-TψC interaction region of the viral RNA results in selection of largely wild-type sequences after five rounds of infection. Also recovered are mutant viruses that maintain their ability to base pair with the TψC loop of the tRNATrp. To prove this interaction is specific to the tRNA primer, we constructed a second library, in which we altered the PBS to anneal to tRNAPro, while simultaneously randomizing the viral RNA U5-TψC region. After five rounds of infection, the consensus sequence 5′-GPuPuCPy-3′ emerged, which is complementary to the 5′-GGTTC-3′ sequence found in the TψC loop of tRNAPro. These observations confirm the importance of the U5-TψC interaction in vivo.

Sign in / Sign up

Export Citation Format

Share Document