scholarly journals The Human Immunodeficiency Virus Type 1 TAR RNA Upper Stem-Loop Plays Distinct Roles in Reverse Transcription and RNA Packaging

2000 ◽  
Vol 74 (12) ◽  
pp. 5639-5646 ◽  
Author(s):  
David Harrich ◽  
C. William Hooker ◽  
Emma Parry
Keyword(s):  
Reverse Transcription ◽  
Transcription Initiation ◽  
Loop Structure ◽  
Rna Packaging ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Immunodeficiency Virus ◽  
Tar Rna ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) RNA genome is flanked by a repeated sequence (R) that is required for HIV-1 replication. The first 57 nucleotides of R form a stable stem-loop structure called the transactivation response element (TAR) that can interact with the virally encoded transcription activator protein, Tat, to promote high levels of gene expression. Recently, we demonstrated that TAR is also important for efficient HIV-1 reverse transcription, since HIV-1 mutated in the upper stem-loop of TAR showed a reduced ability both to initiate and to complete reverse transcription. We have analyzed a series of HIV-1 mutant viruses to better defined the structural or sequence elements required for natural endogenous reverse transcription and packaging of virion RNA. Our results indicate that the requirement for TAR in reverse transcription is conformation dependent, since mutants with mutations that alter the upper stem-loop orientation are defective for reverse transcription initiation and have minor defects in RNA packaging. In contrast, TAR mutations that allowed the formation of alternative upper stem-loop structure greatly reduced RNA packaging but did not affect reverse transcription efficiency. These results are consistent with direct involvement of the upper stem-loop structure in packaging of genomic RNA and suggest that the TAR RNA stem-loop from nucleotide +18 to +42 interacts with other components of the reverse transcription initiation complex to promote efficient reverse transcription.

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

2003 ◽  
Vol 77 (7) ◽  
pp. 4060-4069 ◽  
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.

scholarly journals Characterization of the Jembrana Disease Virustat Gene and the cis- andtrans-Regulatory Elements in Its Long Terminal Repeats

1999 ◽  
Vol 73 (1) ◽  
pp. 658-666 ◽  
Author(s):  
Hexin Chen ◽  
Graham Wilcox ◽  
Gde Kertayadnya ◽  
Charles Wood
Keyword(s):  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Bovine Immunodeficiency Virus ◽  
Loop Structure ◽  
Nucleotide Substitutions ◽  
Stem Loop ◽  
Loop Region ◽  
Stem Loop Structure ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Jembrana disease virus (JDV) is a newly identified bovine lentivirus that is closely related to the bovine immunodeficiency virus (BIV). JDV contains a tat gene, encoded by two exons, which has potent transactivation activity. Cotransfection of the JDVtat expression plasmid with the JDV promoter chloramphenicol acetyltransferase (CAT) construct pJDV-U3R resulted in a substantial increase in the level of CAT mRNA transcribed from the JDV long terminal repeat (LTR) and a dramatic increase in the CAT protein level. Deletion analysis of the LTR sequences showed that sequences spanning nucleotides −68 to +53, including the TATA box and the predicted first stem-loop structure of the predicted Tat response element (TAR), were required for efficient transactivation. The results, derived from site-directed mutagenesis experiments, suggested that the base pairing in the stem of the first stem-loop structure in the TAR region was important for JDV Tat-mediated transactivation; in contrast, nucleotide substitutions in the loop region of JDV TAR had less effect. For the JDV LTR, upstream sequences, from nucleotide −196 and beyond, as well as the predicted secondary structures in the R region, may have a negative effect on basal JDV promoter activity. Deletion of these regions resulted in a four- to fivefold increase in basal expression. The JDV Tat is also a potent transactivator of other animal and primate lentivirus promoters. It transactivated BIV and human immunodeficiency virus type 1 (HIV-1) LTRs to levels similar to those with their homologous Tat proteins. In contrast, HIV-1 Tat has minimal effects on JDV LTR expression, whereas BIV Tat moderately transactivated the JDV LTR. Our study suggests that JDV may use a mechanism of transactivation similar but not identical to those of other animal and primate lentiviruses.

scholarly journals Genetic Analysis of a Unique Human Immunodeficiency Virus Type 1 (HIV-1) with a Primer Binding Site Complementary to tRNAMet Supports a Role for U5-PBS Stem-Loop RNA Structures in Initiation of HIV-1 Reverse Transcription

1999 ◽  
Vol 73 (3) ◽  
pp. 1818-1827 ◽  
Author(s):  
Sang-Moo Kang ◽  
Casey D. Morrow
Keyword(s):  
Human Immunodeficiency Virus ◽  
In Vitro Culture ◽  
Reverse Transcription ◽  
Primer Binding Site ◽  
Rna Structures ◽  
Stem Loop ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) exclusively uses tRNA3 Lys to initiate reverse transcription. A novel HIV-1 mutant which stably utilizes tRNAMet rather than tRNA3 Lys as a primer was previously identified [HXB2(Met-AC] (S.-M. Kang, Z. Zhang, and C. D. Morrow, J. Virol. 71:207–217, 1997). Comparison of RNA secondary structures of the unique sequence (U5)-primer binding site (PBS) viral RNA genome alone or complexed with tRNAMet of HXB2(Met-AC) revealed structural motifs in common with the U5-PBS of the wild-type virus. In the current study, mutations were constructed to alter the U5-PBS structure and disrupt the U5-PBS-tRNAMet interaction of the virus derived from HXB2(Met-AC). All of the mutant viruses were infectious following transfection and coculture with SupT1 cells. Analysis of the initiation of reverse transcription revealed that some of the mutants were impaired compared to HXB2(Met-AC). The genetic stability of the PBS from each virus was determined following in vitro culture. Two mutant proviral constructs, one predicted to completely disrupt the stem-loop structure in U5 and the other predicted to destabilize contact regions of U5 with tRNAMet, reverted back to contain a PBS complementary to tRNA3 Lys. All other mutants maintained a PBS complementary to tRNAMetafter in vitro culture, although all contained multiple nucleotide substitutions within the U5-PBS from the starting proviral clones. Most interestingly, a viral mutant containing a 32-nucleotide deletion between nucleotides 142 and 173, encompassing regions in U5 which interact with tRNAMet, maintained a PBS complementary to tRNAMet following in vitro culture. All of the proviral clones recovered from this mutant, however, contained an additional 19-nucleotide insertion in U5. RNA modeling of the U5-PBS from this mutant demonstrated that the additional mutations present in U5 following culture restored RNA structures similar to those modeled from HXB2(Met-AC). These results provide strong genetic evidence that multiple sequence and structural elements in U5 in addition to the PBS are involved in the interaction with the tRNA used for initiation of reverse transcription.

scholarly journals Impact of Human Immunodeficiency Virus Type 1 RNA Dimerization on Viral Infectivity and of Stem-Loop B on RNA Dimerization and Reverse Transcription and Dissociation of Dimerization from Packaging

2000 ◽  
Vol 74 (12) ◽  
pp. 5729-5735 ◽  
Author(s):  
Ni Shen ◽  
Louis Jetté ◽  
Chen Liang ◽  
Mark A. Wainberg ◽  
Michael Laughrea
Keyword(s):  
Reverse Transcription ◽  
Viral Infectivity ◽  
Stem Loop ◽  
Rna Dimerization ◽  
Reduced Genome ◽  
Immunodeficiency Virus ◽  
Genome Dimerization ◽  
Hiv 1 ◽  
Kissing Loop

ABSTRACT The kissing-loop domain (KLD) encompasses a stem-loop, named kissing-loop or dimerization initiation site (DIS) hairpin (nucleotides [nt] 248 to 270 in the human immunodeficiency virus type 1 strains HIV-1Lai and HIV-1Hxb2), seated on top of a 12-nt stem-internal loop called stem-loop B (nt 243 to 247 and 271 to 277). Destroying stem-loop B reduced genome dimerization by ∼50% and proviral DNA synthesis by ∼85% and left unchanged the dissociation temperature of dimeric genomic RNA. The most affected step of reverse transcription was plus-strand DNA transfer, which was reduced by ∼80%. Deleting nt 241 to 256 or 200 to 256 did not reduce genome dimerization significantly more than the destruction of stem-loop B or the DIS hairpin. We conclude that the KLD is nonmodular: mutations in stem-loop B and in the DIS hairpin have similar effects on genome dimerization, reverse transcription, and encapsidation and are also “nonadditive”; i.e., a larger deletion spanning both of these structures has the same effects on genome dimerization and encapsidation as if stem-loop B strongly impacted DIS hairpin function and vice versa. A C258G transversion in the palindrome of the kissing-loop reduced genome dimerization by ∼50% and viral infectivity by ∼1.4 log. Two mutations, CGCG261→UUAA261 (creating a weaker palindrome) and a Δ241–256 suppressor mutation, were each able to reduce genome dimerization but leave genome packaging unaffected.

scholarly journals The Interaction of APOBEC3G with Human Immunodeficiency Virus Type 1 Nucleocapsid Inhibits tRNA3Lys Annealing to Viral RNA

2007 ◽  
Vol 81 (20) ◽  
pp. 11322-11331 ◽  
Author(s):  
Fei Guo ◽  
Shan Cen ◽  
Meijuan Niu ◽  
Yiliang Yang ◽  
Robert J. Gorelick ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Transcription Initiation ◽  
Rna Binding ◽  
Viral Rna ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) containing human APOBEC3G (hA3G) has a reduced ability to produce viral DNA in newly infected cells. At least part of this hA3G-facilitated inhibition is due to a cytidine deamination-independent reduction in the ability to initiate reverse transcription. HIV-1 nucleocapsid (NCp7) is required both for the incorporation of hA3G into virions and for the annealing between viral RNA and tRNA3 Lys, the primer tRNA for reverse transcription. Herein we present evidence that the interaction of hA3G with nucleocapsid is required for the inhibition of reverse transcription initiation. A tRNA3 Lys priming complex was produced in vitro by the NCp7-facilitated annealing of tRNA3 Lys to synthetic viral RNA in the absence or presence of hA3G. The effect of hA3G on the annealing of tRNA3 Lys to viral RNA and the ability of tRNA3 Lys to initiate reverse transcription was measured. Our results show the following. (i) Electrophoretic band shift and primer binding site assays show that hA3G reduces the annealing of tRNA3 Lys 44 and 60%, respectively, but does not disrupt the annealed complex once formed. (ii) hA3G inhibits tRNA3 Lys priming 70 to 80%. (iii) Inhibition of tRNA3 Lys priming by hA3G requires an interaction between hA3G and NCp7 during annealing. Thus, annealing of tRNA3 Lys is insensitive to hA3G inhibition when facilitated by a zinc finger mutant of NCp7 unable to interact with hA3G. NCp7-independent annealing of DNA to viral RNA also is insensitive to hA3G inhibition. These results indicate that hA3G does not sterically block tRNA3 Lys annealing by binding to viral RNA. Annealing and priming are not affected by another RNA binding protein, QKI-6.

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

1999 ◽  
Vol 73 (1) ◽  
pp. 101-109 ◽  
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.

scholarly journals Kinetics of Human Immunodeficiency Virus Type 1 Decay following Entry into Resting CD4+ T Cells

2005 ◽  
Vol 79 (4) ◽  
pp. 2199-2210 ◽  
Author(s):  
Yan Zhou ◽  
Haili Zhang ◽  
Janet D. Siliciano ◽  
Robert F. Siliciano
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Human Immunodeficiency Virus Type ◽  
Reverse Transcription ◽  
Half Life ◽  
Immunodeficiency Virus ◽  
Kinetics Of ◽  
Hiv 1

ABSTRACT In untreated human immunodeficiency virus type 1 (HIV-1) infection, most viral genomes in resting CD4+ T cells are not integrated into host chromosomes. This unintegrated virus provides an inducible latent reservoir because cellular activation permits integration, virus gene expression, and virus production. It remains controversial whether HIV-1 is stable in this preintegration state. Here, we monitored the fate of HIV-1 in resting CD4+ cells by using a green fluorescent protein (GFP) reporter virus carrying an X4 envelope. After virus entry into resting CD4+ T cells, both rescuable virus gene expression, visualized with GFP, and rescuable virion production, assessed by p24 release, decayed with a half-life of 2 days. In these cells, reverse transcription goes to completion over 2 to 3 days, and 50% of the viruses that have entered undergo functional decay before reverse transcription is complete. We distinguished two distinct but closely related factors contributing to loss of rescuable virus. First, some host cells undergo virus-induced apoptosis upon viral entry, thereby reducing the amount of rescuable virus. Second, decay processes directly affecting the virus both before and after the completion of reverse transcription contribute to the loss of rescuable virus. The functional half-life of full-length, integration-competent reverse transcripts is only 1 day. We propose that rapid intracellular decay processes compete with early steps in viral replication in infected CD4+ T cells. Decay processes dominate in resting CD4+ T cells as a result of the slow kinetics of reverse transcription and blocks at subsequent steps. Therefore, the reservoir of unintegrated HIV-1 in recently infected resting CD4+ T cells is highly labile.

Sign in / Sign up

Export Citation Format

Share Document