Identification of Critical Elements in the tRNA Acceptor Stem and TΨC Loop Necessary for Human Immunodeficiency Virus Type 1 Infectivity

ABSTRACT A mutant human immunodeficiency virus type 1 (HIV-1) with a primer binding site (PBS) complementary to yeast tRNAPhe(psHIV-Phe), which relies on exogenous yeast tRNAPhe as reverse transcription primer, was used to investigate elements in the tRNA acceptor stem and TΨC stem-loop required for the tRNA primer selection and use in HIV-1 replication. tRNAPhe mutants with two- or four-nucleotide deletions in the 3′ end retained the capacity to complement replication of psHIV-Phe. tRNAPhemutants with an extended 5′ end had reduced capacity for complementation, which could be restored by extension of the 3′ end of these tRNAPhe mutants with sequences complementary to the HIV-1 U5 region. Further analysis of mutations in the acceptor stem of tRNAPhe suggested that an intact acceptor stem RNA structure is important for complementation. Analysis of single-nucleotide changes in the TΨC stem-loop of tRNAPherevealed an unexpected, essential role of this region for rescue of psHIV-Phe.

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Possible Role of Dimerization in Human Immunodeficiency Virus Type 1 Genome RNA Packaging

Journal of Virology ◽

10.1128/jvi.77.7.4060-4069.2003 ◽

2003 ◽

Vol 77 (7) ◽

pp. 4060-4069 ◽

Cited By ~ 44

Author(s):

Jun-Ichi Sakuragi ◽

Shigeharu Ueda ◽

Aikichi Iwamoto ◽

Tatsuo Shioda

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Rna Packaging ◽

Dimer Formation ◽

Stem Loop ◽

Immunodeficiency Virus ◽

Lower Stem ◽

Hiv 1

ABSTRACT The dimer initiation site/dimer linkage sequence (DIS/DLS) region in the human immunodeficiency virus type 1 (HIV-1) RNA genome is suggested to play important roles in various steps of the virus life cycle. However, due to the presence of a putative DIS/DLS region located within the encapsidation signal region (E/psi), it is difficult to perform a mutational analysis of DIS/DLS without affecting the packaging of RNA into virions. Recently, we demonstrated that duplication of the DIS/DLS region in viral RNA caused the production of partially monomeric RNAs in virions, indicating that the region indeed mediated RNA-RNA interaction. We utilized this system to assess the precise location of DIS/DLS in the 5′ region of the HIV-1 genome with minimum effect on RNA packaging. We found that the entire lower stem of the U5/L stem-loop was required for packaging, whereas the region important for dimer formation was only 10 bases long within the lower stem of the U5/L stem-loop. The R/U5 stem-loop was required for RNA packaging but was completely dispensable for dimer formation. The SL1 lower stem was important for both dimerization and packaging, but surprisingly, deletion of the palindromic sequence at the top of the loop only partially affected dimerization. These results clearly indicated that the E/psi of HIV-1 is much larger than the DIS/DLS and that the primary DIS/DLS is completely included in the E/psi. Therefore, it is suggested that RNA dimerization is a part of RNA packaging, which requires multiple steps.

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The TAR Hairpin of Human Immunodeficiency Virus Type 1 Can Be Deleted When Not Required for Tat-Mediated Activation of Transcription

Journal of Virology ◽

10.1128/jvi.00392-07 ◽

2007 ◽

Vol 81 (14) ◽

pp. 7742-7748 ◽

Cited By ~ 38

Author(s):

Atze T. Das ◽

Alex Harwig ◽

Martine M. Vrolijk ◽

Ben Berkhout

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Stem Loop ◽

Activation Of Transcription ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) RNA genome contains a terminal repeat (R) region that encodes the transacting responsive (TAR) hairpin, which is essential for Tat-mediated activation of gene expression. TAR has also been implicated in several other processes during viral replication, including translation, dimerization, packaging, and reverse transcription. However, most studies in which replication of TAR-mutated viruses was analyzed were complicated by the dominant negative effect of the mutations on transcription. We therefore used an HIV-1 variant that does not require TAR for transcription to reinvestigate the role of TAR in HIV-1 replication. We demonstrate that this virus can replicate efficiently upon complete deletion of TAR. Furthermore, evolution of a TAR-deleted variant in long-term cultures indicates that HIV-1 requires a stable stem-loop structure at the start of the viral transcripts in which the 5′-terminal nucleotides are base paired. This prerequisite for efficient replication can be fulfilled by the TAR hairpin but also by unrelated stem-loop structures. We therefore conclude that TAR has no essential function in HIV-1 replication other than to accommodate Tat-mediated activation of transcription.

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Genetic Dissociation of the Encapsidation and Reverse Transcription Functions in the 5′ R Region of Human Immunodeficiency Virus Type 1

Journal of Virology ◽

10.1128/jvi.73.1.101-109.1999 ◽

1999 ◽

Vol 73 (1) ◽

pp. 101-109 ◽

Cited By ~ 41

Author(s):

Jared L. Clever ◽

Daniel A. Eckstein ◽

Tristram G. Parslow

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Stem Loop ◽

Immunodeficiency Virus ◽

R Region ◽

Compensatory Mutations ◽

Hiv 1

ABSTRACT The efficient packaging of genomic RNA into virions of human immunodeficiency virus type 1 (HIV-1) is directed bycis-acting encapsidation signals, which have been mapped to particular RNA stem-loop structures near the 5′ end of the genome. Earlier studies have shown that three such stem-loops, located adjacent to the major 5′ splice donor, are required for optimal packaging; more recent reports further suggest a requirement for the TAR and poly(A) hairpins of the 5′ R region. In the present study, we have compared the phenotypes that result from mutating these latter elements in the HIV-1 provirus. Using a single-round infectivity assay, we find that mutations which disrupt base pairing in either the TAR or poly(A) stems cause profound defects in both packaging and viral replication. Decreased genomic packaging in a given mutant was always accompanied by increased packaging of spliced viral RNAs. Compensatory mutations that restored base pairing also restored encapsidation, indicating that the secondary structures of the TAR and poly(A) stems, rather than their primary sequences, are important for packaging activity. Despite having normal RNA contents, however, viruses with compensatory mutations at the base of the TAR stem were severely replication defective, owing to a defect in proviral DNA synthesis. Our findings thus confirm that the HIV-1 TAR stem-loop is required for at least three essential viral functions (transcriptional activation, RNA packaging, and reverse transcription) and reveal that its packaging and reverse transcription activities can be dissociated genetically by mutations at the base of the TAR stem.

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