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

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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Dissociation of Genome Dimerization from Packaging Functions and Virion Maturation of Human Immunodeficiency Virus Type 1

Journal of Virology ◽

10.1128/jvi.76.3.959-967.2002 ◽

2002 ◽

Vol 76 (3) ◽

pp. 959-967 ◽

Cited By ~ 31

Author(s):

Jun-ichi Sakuragi ◽

Aikichi Iwamoto ◽

Tatsuo Shioda

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Viral Rna ◽

Rna Packaging ◽

Virus Particles ◽

Immunodeficiency Virus ◽

Rna Interaction ◽

Hiv 1

ABSTRACT The dimer initiation site/dimer linkage sequence (DIS/DLS) region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is thought to play important roles at various stages of the virus life cycle. Recently we showed that the DIS/DLS region affects RNA-RNA interaction in intact virus particles, by demonstrating that duplication of the region in viral RNA caused the production of virus particles containing partially monomeric RNAs. We have extended this finding and succeeded for the first time in creating mutant particles which contain only monomeric RNAs without modifying any viral proteins. In terms of RNA encapsidation ability, virion density, and protein processing, the mutant particles were comparable to wild-type particles. The level of production of viral DNA by the mutant virus construct in infected cells was also comparable to that of the constructs that produced exclusively dimeric RNA, indicating that monomeric viral RNA could be the template for strand transfer. These results indicated that the RNA dimerization of HIV-1 could be separated from viral RNA packaging and was not absolutely required for RNA packaging, virion maturation, and reverse transcription.

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A Riboswitch Regulates RNA Dimerization and Packaging in Human Immunodeficiency Virus Type 1 Virions

Journal of Virology ◽

10.1128/jvi.78.19.10814-10819.2004 ◽

2004 ◽

Vol 78 (19) ◽

pp. 10814-10819 ◽

Cited By ~ 65

Author(s):

Marcel Ooms ◽

Hendrik Huthoff ◽

Rodney Russell ◽

Chen Liang ◽

Ben Berkhout

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Large Set ◽

Rna Packaging ◽

Rna Dimerization ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The genome of retroviruses, including human immunodeficiency virus type 1 (HIV-1), consists of two identical RNA strands that are packaged as noncovalently linked dimers. The core packaging and dimerization signals are located in the downstream part of the untranslated leader of HIV-1 RNA—the Ψ and the dimerization initiation site (DIS) hairpins. The HIV-1 leader can adopt two alternative conformations that differ in the presentation of the DIS hairpin and consequently in their ability to dimerize in vitro. The branched multiple-hairpin (BMH) structure folds the poly(A) and DIS hairpins, but these domains are base paired in a long distance interaction (LDI) in the most stable LDI conformation. This LDI-BMH riboswitch regulates RNA dimerization in vitro. It was recently shown that the Ψ hairpin structure is also presented differently in the LDI and BMH structures. Several detailed in vivo studies have indicated that sequences throughout the leader RNA contribute to RNA packaging, but how these diverse mutations affect the packaging mechanism is not known. We reasoned that these effects may be due to a change in the LDI-BMH equilibrium, and we therefore reanalyzed the structural effects of a large set of leader RNA mutations that were presented in three previous studies (J. L. Clever, D. Mirandar, Jr., and T. G. Parslow, J. Virol. 76:12381-12387, 2002; C. Helga-Maria, M. L. Hammarskjold, and D. Rekosh, J. Virol. 73:4127-4135, 1999; R. S. Russell, J. Hu, V. Beriault, A. J. Mouland, M. Laughrea, L. Kleiman, M. A. Wainberg, and C. Liang, J. Virol. 77:84-96, 2003). This analysis revealed a strict correlation between the status of the LDI-BMH equilibrium and RNA packaging. Furthermore, a correlation is apparent between RNA dimerization and RNA packaging, and these processes may be coordinated by the same LDI-BMH riboswitch mechanism.

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Context-Dependent Phenotype of a Human Immunodeficiency Virus Type 1 Nucleocapsid Mutation

Journal of Virology ◽

10.1128/jvi.75.15.7193-7197.2001 ◽

2001 ◽

Vol 75 (15) ◽

pp. 7193-7197 ◽

Cited By ~ 6

Author(s):

Andrea Cimarelli ◽

Jeremy Luban

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Rna Packaging ◽

Solution Structures ◽

Immunodeficiency Virus ◽

Packaging Efficiency ◽

Viral Genomic Rna ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) nucleocapsid mutation R10A/K11A abolishes viral replication when present in proviral clone HIV-1HXB-2, but it was found to have minimal effect on replication of the closely related HIV-1NL4-3. Functional mapping demonstrated that a nonconservative amino acid change at nucleocapsid residue 24 (threonine in HIV-1HXB-2, isoleucine in HIV-1NL4-3) is the major determinant of the different R10A/K11A phenotypes in these two proviruses. Threonine-isoleucine exchanges appear to modify the R10A/K11A phenotype via effects on virion RNA-packaging efficiency. The improved packaging seen with hydrophobic isoleucine is consistent with solution structures localizing this residue to a hydrophobic pocket that contacts guanosine bases in viral genomic RNA stem-loops critical for packaging.

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Infectious Molecular Clones with the Nonhomologous Dimer Initiation Sequences Found in Different Subtypes of Human Immunodeficiency Virus Type 1 Can Recombine and Initiate a Spreading Infection In Vitro

Journal of Virology ◽

10.1128/jvi.72.5.3991-3998.1998 ◽

1998 ◽

Vol 72 (5) ◽

pp. 3991-3998 ◽

Cited By ~ 52

Author(s):

Daniel C. St. Louis ◽

Deanna Gotte ◽

Eric Sanders-Buell ◽

David W. Ritchey ◽

Mika O. Salminen ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Rna Packaging ◽

Immunodeficiency Virus ◽

Rna Dimer ◽

Initiation Sequence ◽

Hiv 1

ABSTRACT Recombinant forms of human immunodeficiency virus type 1 (HIV-1) have been shown to be of major importance in the global AIDS pandemic. Viral RNA dimer formation mediated by the dimerization initiation sequence (DIS) is believed to be essential for viral genomic RNA packaging and therefore for RNA recombination. Here, we demonstrate that HIV-1 recombination and replication are not restricted by variant DIS loop sequences. Three DIS loop forms found among HIV-1 isolates, DIS (CG), DIS (TA), and DIS (TG), when introduced into deletion mutants of HIV-1 recombined efficiently, and the progeny virions replicated with comparable kinetics. A fourth DIS loop form, containing an artificial AAAAAA sequence disrupting the putative DIS loop-loop interactions [DIS (A6)], supported efficient recombination with DIS loop variants; however, DIS (A6) progeny virions exhibited a modest replication disadvantage in mixed cultures. Our studies indicate that the nonhomologous DIS sequences found in different HIV-1 subtypes are not a primary obstacle to intersubtype recombination.

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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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An Intact TAR Element and Cytoplasmic Localization Are Necessary for Efficient Packaging of Human Immunodeficiency Virus Type 1 Genomic RNA

Journal of Virology ◽

10.1128/jvi.73.5.4127-4135.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 4127-4135 ◽

Cited By ~ 51

Author(s):

C. Helga-Maria ◽

Marie-Louise Hammarskjöld ◽

David Rekosh

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Splice Site ◽

Genomic Rna ◽

Rna Packaging ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Stem Structure

ABSTRACT Although most reports defining the human immunodeficiency virus type 1 (HIV-1) genomic RNA packaging signal have focused on the region downstream of the major 5′ splice site, others have suggested that sequences upstream of the splice site may also play an important role. In this study we have directly examined the role played by the HIV-1 TAR region in RNA packaging. For these experiments we used a proviral expression system that is largely independent of Tat for transcriptional activation. This allowed us to create constructs that efficiently expressed RNAs carrying mutations in TAR and to determine the ability of these RNAs to be packaged. Our results indicate that loss of sequences in TAR significantly reduce the ability of a viral RNA to be packaged. The requirement for TAR sequences in RNA packaging was further examined by using a series of missense mutations positioned throughout the entire TAR structure. TAR mutations previously shown to influence Tat transactivation, such as G31U in the upper loop region or UCU to AAG in the bulge (nucleotides [nt] 22 to 24), failed to have any effect on RNA packaging. Mutations which disrupted the portion of the TAR stem immediately below the bulge also had little effect. In contrast, dramatic effects on RNA packaging were observed with constructs containing mutations in the lower portion of the TAR stem. Point mutations which altered nt 5 to 9, 10 to 15, 44 to 49, or 50 to 54 all reduced RNA packaging 11- to 25-fold. However, compensatory double mutations which restored the stem structure were able to restore packaging. These results indicate that an intact lower stem structure, rather than a specific sequence, is required for RNA packaging. Our results also showed that RNA molecules retained within the nucleus cannot be packaged, unless they are transported to the cytoplasm by either Rev/Rev response element or the Mason-Pfizer monkey virus constitutive transport element.

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

Journal of Virology ◽

10.1128/jvi.75.10.4902-4906.2001 ◽

2001 ◽

Vol 75 (10) ◽

pp. 4902-4906 ◽

Cited By ~ 8

Author(s):

Qin Yu ◽

Casey D. Morrow

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Stem Loop ◽

Yeast Trna ◽

Acceptor Stem ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Trna Acceptor

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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