Direct structural evidence for formation of a stem-loop structure involved in ribosomal frameshifting in human immunodeficiency virus type 1

ABSTRACT Human immunodeficiency virus type 1 uses ribosomal frameshifting for translation of the Gag-Pol polyprotein. Frameshift activities are thought to be tightly regulated. Analysis of gag p1 sequences from 270 plasma virions identified in 64% of the samples the occurrence of polymorphism that could lead to changes in thermodynamic stability of the stem-loop. Expression in Saccharomyces cerevisiae of p1-β-galactosidase fusion proteins from 10 representative natural stem-loop variants and three laboratory mutant constructs (predicted the thermodynamic stability [ΔG°] ranging from −23.0 to −4.3 kcal/mol) identified a reduction in frameshift activity of 13 to 67% compared with constructs with the wild-type stem-loop (ΔG°, −23.5 kcal/mol). Viruses carrying stem-loops associated with greater than 60% reductions in frameshift activity presented profound defects in viral replication. In contrast, viruses with stem-loop structures associated with 16 to 42% reductions in frameshift efficiency displayed no significant viral replication deficit.

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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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Novel Gag-Pol Frameshift Site in Human Immunodeficiency Virus Type 1 Variants Resistant to Protease Inhibitors

Journal of Virology ◽

10.1128/jvi.72.7.6146-6150.1998 ◽

1998 ◽

Vol 72 (7) ◽

pp. 6146-6150 ◽

Cited By ~ 52

Author(s):

Louise Doyon ◽

Catherine Payant ◽

Léa Brakier-Gingras ◽

Daniel Lamarre

Keyword(s):

Human Immunodeficiency Virus ◽

Protease Inhibitors ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Cleavage Site ◽

Messenger Rna ◽

Site Mutation ◽

Ribosomal Frameshifting ◽

Immunodeficiency Virus

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease inhibitors have been shown to contain a mutation in the p1/p6 Gag precursor cleavage site. At the messenger RNA level, this mutation generates a U UUU UUU sequence that is reminiscent of the U UUU UUA sequence required for ribosomal frameshifting and Gag-Pol synthesis. To test whether the p1/p6 cleavage site mutation was generating a novel frameshift site, HIV sequences were inserted in translation vectors containing a chloramphenicol acetyltransferase (CAT) reporter gene requiring −1 frameshifting for expression. All sequences containing the original HIV frameshift site supported the synthesis of CAT but expression was increased 3- to 11-fold in the presence of the mutant p1/p6 sequence. When the original frameshift site was abolished by mutation, expression remained unchanged when using constructs containing the mutant p1/p6 sequence, whereas it was decreased 2- to 4.5-fold when using wild-type p1/p6 constructs. Similarly, when introduced into HIV molecular clones, the p1/p6 mutant sequence supported Gag-Pol synthesis and protease activity in the absence of the original frameshift site, indicating that this sequence could also promote ribosomal frameshifting in virus-expressing cells.

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Deletion of stem-loop 3 is compensated by second-site mutations within the Gag protein of human immunodeficiency virus type 1

Virology ◽

10.1016/s0042-6822(03)00405-7 ◽

2003 ◽

Vol 314 (1) ◽

pp. 221-228 ◽

Cited By ~ 11

Author(s):

Liwei Rong ◽

Rodney S Russell ◽

Jing Hu ◽

Michael Laughrea ◽

Mark A Wainberg ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Stem Loop ◽

Gag Protein ◽

Immunodeficiency Virus

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Inhibition of Human Immunodeficiency Virus Type-1 env Expression by C-5 Propyne Oligonucleotides Specific for Rev-Response Element Stem-Loop V

Biochemistry ◽

10.1021/bi00194a002 ◽

1994 ◽

Vol 33 (28) ◽

pp. 8391-8398 ◽

Cited By ~ 41

Author(s):

Steven D. Fenster ◽

Richard W. Wagner ◽

Brian C. Froehler ◽

Daniel J. Chin

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Response Element ◽

Stem Loop ◽

Rev Response Element ◽

Immunodeficiency Virus

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The Human Immunodeficiency Virus Type 1 Ribosomal Frameshifting Site Is an Invariant Sequence Determinant and an Important Target for Antiviral Therapy

Journal of Virology ◽

10.1128/jvi.78.4.2082-2087.2004 ◽

2004 ◽

Vol 78 (4) ◽

pp. 2082-2087 ◽

Cited By ~ 51

Author(s):

Preetha Biswas ◽

Xi Jiang ◽

Annmarie L. Pacchia ◽

Joseph P. Dougherty ◽

Stuart W. Peltz

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Sequence Comparison ◽

Ribosomal Frameshifting ◽

Important Target ◽

Immunodeficiency Virus ◽

Sequence Determinant ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) utilizes a distinctive form of gene regulation as part of its life cycle, termed programmed −1 ribosomal frameshifting, to produce the required ratio of the Gag and Gag-Pol polyproteins. We carried out a sequence comparison of 1,000 HIV-1 sequences at the slippery site (UUUUUUA) and found that the site is invariant, which is somewhat surprising for a virus known for its variability. This prompted us to prepare a series of mutations to examine their effect upon frameshifting and viral infectivity. Among the series of mutations were changes of the HIV-1 slippery site to those effectively utilized by other viruses, because such mutations would be anticipated to have a relatively mild effect upon frameshifting. The results demonstrate that any change to the slippery site reduced frameshifting levels and also dramatically inhibited infectivity. Because ribosomal frameshifting is essential for HIV-1 replication and it is surprisingly resistant to mutation, modulation of HIV-1 frameshifting efficiency potentially represents an important target for the development of novel antiviral therapeutics.

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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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