scholarly journals Multiple molecular determinants for retrotransposition in a primer tRNA.

1995 ◽  
Vol 15 (1) ◽  
pp. 217-226 ◽  
Author(s):  
J B Keeney ◽  
K B Chapman ◽  
V Lauermann ◽  
D F Voytas ◽  
S U Aström ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Reverse Transcription ◽  
Primer Binding Site ◽  
Specific Interactions ◽  
Acceptor Stem ◽  
Transcription Process ◽  
Molecular Determinants ◽  
Genetic Assay ◽  
Strong Stop ◽  
Interspecies Hybrid

Retroviruses and long terminal repeat-containing retroelements use host-encoded tRNAs as primers for the synthesis of minus strong-stop DNA, the first intermediate in reverse transcription of the retroelement RNA. Usually, one or more specific tRNAs, including the primer, are selected and packaged within the virion. The reverse transcriptase (RT) interacts with the primer tRNA and initiates DNA synthesis. The structural and sequence features of primer tRNAs important for these specific interactions are poorly understood. We have developed a genetic assay in which mutants of tRNA(iMet), the primer for the Ty1 retrotransposon of Saccharomyces cerevisiae, can be tested for the ability to serve as primers in the reverse transcription process. This system allows any tRNA mutant to be tested, regardless of its ability to function in the initiation of protein synthesis. We find that mutations in the T psi C loop and the acceptor stem regions of the tRNA(iMet) affect transposition most severely. Conversely, mutations in the anticodon region have only minimal effects on transposition. Further study of the acceptor stem and other mutants demonstrates that complementarity to the element primer binding site is a necessary but not sufficient requirement for effective tRNA priming. Finally, we have used interspecies hybrid initiator tRNA molecules to implicate nucleotides in the D arm as additional recognition determinants. Ty3 and Ty1, two very distantly related retrotransposons, require similar molecular determinants in this primer tRNA for transposition.

scholarly journals Interactions between Ty1 Retrotransposon RNA and the T and D Regions of the tRNAiMet Primer Are Required for Initiation of Reverse Transcription In Vivo

1998 ◽  
Vol 18 (2) ◽  
pp. 799-806 ◽  
Author(s):  
S. Friant ◽  
T. Heyman ◽  
A. S. Byström ◽  
M. Wilhelm ◽  
F. X. Wilhelm
Keyword(s):  
Reverse Transcription ◽  
Primer Binding Site ◽  
Minus Strand ◽  
Genomic Rna ◽  
Complementary Sequences ◽  
Compensatory Mutations ◽  
Ty1 Retrotransposon ◽  
Ty1 Retrotransposition ◽  
Strong Stop

ABSTRACT Reverse transcription of the Saccharomyces cerevisiaeTy1 retrotransposon is primed by tRNAi Met base paired to the primer binding site (PBS) near the 5′ end of Ty1 genomic RNA. The 10-nucleotide PBS is complementary to the last 10 nucleotides of the acceptor stem of tRNAi Met. A structural probing study of the interactions between the Ty1 RNA template and the tRNAi Met primer showed that besides interactions between the PBS and the 3′ end of tRNAi Met, three short regions of Ty1 RNA, named boxes 0, 1, and 2.1, interact with the T and D stems and loops of tRNAi Met. To determine if these sequences are important for the reverse transcription pathway of the Ty1 retrotransposon, mutant Ty1 elements and tRNAi Metwere tested for the ability to support transposition. We show that the Ty1 boxes and the complementary sequences in the T and D stems and loops of tRNAi Met contain bases that are critical for Ty1 retrotransposition. Disruption of 1 or 2 bp between tRNAi Met and box 0, 1, or 2.1 dramatically decreases the level of transposition. Compensatory mutations which restore base pairing between the primer and the template restore transposition. Analysis of the reverse transcription intermediates generated inside Ty1 virus-like particles indicates that initiation of minus-strand strong-stop DNA synthesis is affected by mutations disrupting complementarity between Ty1 RNA and primer tRNAi Met.

scholarly journals An unusual mechanism of self-primed reverse transcription requires the RNase H domain of reverse transcriptase to cleave an RNA duplex.

1996 ◽  
Vol 16 (10) ◽  
pp. 5645-5654 ◽  
Author(s):  
H L Levin
Keyword(s):  
Reverse Transcriptase ◽  
Long Terminal Repeat ◽  
Reverse Transcription ◽  
Rnase H ◽  
Terminal Repeat ◽  
Primer Binding Site ◽  
The Novel ◽  
Double Stranded Rna ◽  
Trna Species ◽  
Strong Stop

The reverse transcription of retroviruses and long terminal repeat-containing retrotransposons requires that tRNA species serve as primers. We recently reported that the long terminal repeat-containing retrotransposon Tf1 is a unique exception in that reverse transcription is independent of tRNA and is instead initiated by a self-priming mechanism. The first 11 bases of the Tf1 transcript fold back and anneal to the primer binding site in a process that results in the priming of minus-strand strong-stop DNA. Data presented here demonstrate that a cleavage occurs between the 11th and 12th bases of the transcript, resulting in the generation of the primer. Mutagenesis experiments presented here indicate that the RNase H domain of the Tf1 reverse transcriptase is required for the cleavage reaction, suggesting that this RNase H may have the novel ability to cleave double-stranded RNA at the end of a duplexed region.

scholarly journals Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity

2020 ◽  
Vol 22 (1) ◽  
pp. 58
Author(s):  
Thomas Gremminger ◽  
Zhenwei Song ◽  
Juan Ji ◽  
Avery Foster ◽  
Kexin Weng ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Potential Interaction ◽  
Viral Rna ◽  
Primer Binding Site ◽  
Rna Integrity ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Extended Interaction ◽  
Hiv 1

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

Thermosensitivity of the Reverse Transcription Process as an Inactivation Mechanism of Lentiviral Vectors

2009 ◽  
Vol 20 (10) ◽  
pp. 1168-1176 ◽  
Author(s):  
M. Carmo ◽  
J.D. Dias ◽  
A. Panet ◽  
A.S. Coroadinha ◽  
M.J.T. Carrondo ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Lentiviral Vectors ◽  
Transcription Process ◽  
Inactivation Mechanism

Viral RNA structural equilibria and their interactions with host proteins

2019 ◽  
Author(s):  
◽  
Samantha Elizabeth Brady
Keyword(s):  
Reverse Transcription ◽  
Rna Structure ◽  
Drug Targets ◽  
Rna Viruses ◽  
Rna Virus ◽  
Protein Translation ◽  
Viral Rna ◽  
Primer Binding Site ◽  
In Vitro Techniques

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Understanding viral RNA structure and how it functions is crucial in elucidating new drug targets. There are many kinds of viruses that utilize RNA as a critical component of their life cycle, such as retroviruses, single-stranded plus or minus sense RNA viruses, and double-stranded RNA viruses. Two viruses that are studied in this thesis are human immunodeficiency virus (HIV), which is a retrovirus, and hepatitis C virus (HCV), which is a single-stranded plus sense RNA virus. It has been previously reported that a human host factor, RNA helicase A (RHA), is packaged into HIV virions by binding to the primer binding site (PBS) segment of the 5'untranslated region in the HIV genomic RNA. We determined RHA is required for efficient reverse transcription prior to capsid uncoating by utilizing cell based and in vitro techniques. It has also been suggested that RHA plays other roles during HIV infection besides reverse transcription. Utilizing NMR, we demonstrated that RHA binds to the monomeric 5'UTR at the bottom of the TAR hairpin, which is different from how it binds during viral packaging. Next, we employed NMR techniques to probe the 3'end of the HCV genome called 3'X. We determined that the 3'X is in structural equilibrium between two states: an open conformation and a closed conformation. These two conformations have been suggested to play a role in minus sense synthesis and viral protein translation, respectively. Taken together, my thesis work has elucidated how many viruses manipulate and utilize their RNA structure to modulate their outcome.

scholarly journals Few Mutations in the 5′ Leader Region Mediate Fitness Recovery of Debilitated Human Immunodeficiency Type 1 Viruses

2005 ◽  
Vol 79 (9) ◽  
pp. 5421-5427 ◽  
Author(s):  
Eloísa Yuste ◽  
Antonio V. Bordería ◽  
Esteban Domingo ◽  
Cecilio López-Galíndez
Keyword(s):  
Reverse Transcription ◽  
Large Population ◽  
Primer Binding Site ◽  
Leader Region ◽  
Coding Regions ◽  
Immunodeficiency Virus ◽  
Large Populations ◽  
Large Virus ◽  
Hiv 1

ABSTRACT Repeated bottleneck passages of RNA viruses result in fitness losses due to the accumulation of deleterious mutations. In contrast, repeated transfers of large virus populations result in exponential fitness increases. Human immunodeficiency virus type 1 (HIV-1) manifested a drastic fitness loss after a limited number of plaque-to-plaque transfers in MT-4 cells. An analysis of the mutations associated with fitness loss in four debilitated clones revealed mutation frequencies in gag that were threefold higher than those in env. We now show an increase in the fitness of the debilitated HIV-1 clones by repeated passages of large populations. An analysis of the entire genomic nucleotide sequences of these populations showed that few mutations, from two to seven per clone, mediated fitness recovery. Eight of the 20 mutations affected coding regions, mainly by the introduction of nonsynonymous mutations (75%). However, most of the mutations accumulated during fitness recovery (12 of 20) were located in the 5′ untranslated leader region of the genome, and more specifically, in the primer binding site (PBS) loop. Two of the viruses incorporated the same mutation in the primer activation signal in the PBS loop, which is critical for the tRNA3 Lys-mediated initiation of reverse transcription. Moreover, 25% of the mutations observed were reversions. This fact, together with the presence of a large proportion of nonsynonymous replacements, may disclose the operation, during large population passages, of strong positive selection for optimal HIV-1 replication, which seems to be primarily affected by binding of the tRNA to the PBS and the initiation of reverse transcription.

How the HIV-1 Nucleocapsid Protein Binds and Destabilises the (−)Primer Binding Site During Reverse Transcription

2008 ◽  
Vol 383 (5) ◽  
pp. 1112-1128 ◽  
Author(s):  
Sarah Bourbigot ◽  
Nick Ramalanjaona ◽  
Christian Boudier ◽  
Gilmar F.J. Salgado ◽  
Bernard P. Roques ◽  
...  
Keyword(s):  
Binding Site ◽  
Reverse Transcription ◽  
Nucleocapsid Protein ◽  
Primer Binding Site ◽  
Hiv 1

scholarly journals Stabilizing Effects of Interruptions on Trinucleotide Repeat Expansions in Saccharomyces cerevisiae

2000 ◽  
Vol 20 (1) ◽  
pp. 173-180 ◽  
Author(s):  
Michael L. Rolfsmeier ◽  
Robert S. Lahue
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Base Pair ◽  
Trinucleotide Repeat ◽  
Primary Factor ◽  
Yeast Cells ◽  
Repeat Expansions ◽  
Yeast Genetic ◽  
Hairpin Formation ◽  
Lagging Strand ◽  
Genetic Assay

ABSTRACT In most trinucleotide repeat (TNR) diseases, the primary factor determining the likelihood of expansions is the length of the TNR. In some diseases, however, stable alleles contain one to three base pair substitutions that interrupt the TNR tract. The unexpected stability of these alleles compared to the frequent expansions of perfect TNRs suggested that interruptions somehow block expansions and that expansions occur only upon loss of at least one interruption. The work in this study uses a yeast genetic assay to examine the mechanism of stabilization conferred by two interruptions of a 25-repeat tract. Expansion rates are reduced up to 90-fold compared to an uninterrupted allele. Stabilization is greatest when the interruption is replicated early on the lagging strand, relative to the rest of the TNR. Although expansions are infrequent, they are often polar, gaining new DNA within the largest available stretch of perfect repeats. Surprisingly, interruptions are always retained and sometimes even duplicated, suggesting that expansion in yeast cells can proceed without loss of the interruption. These findings support a stabilization model in which interruptions contribute in cis to reduce hairpin formation during TNR replication and thus inhibit expansion rates.

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