scholarly journals Identification of a Novel Posttranscriptional Regulatory Element by Using a rev- and RRE-Mutated Human Immunodeficiency Virus Type 1 DNA Proviral Clone as a Molecular Trap

2001 ◽  
Vol 75 (10) ◽  
pp. 4558-4569 ◽  
Author(s):  
Filomena Nappi ◽  
Ralf Schneider ◽  
Andrei Zolotukhin ◽  
Sergey Smulevitch ◽  
Daniel Michalowski ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Regulatory Element ◽  
Mrna Export ◽  
Regulatory Elements ◽  
Control Element ◽  
Rna Transport ◽  
Human Lymphoid Cell ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus (HIV) and all other lentiviruses utilize the essential viral protein Rev, which binds to RRE RNA, to export their unspliced and partially spliced mRNAs from the nucleus. We used a rev- and RRE-defective HIV type 1 (HIV-1) molecular clone in complementation experiments to establish a method for the rapid isolation of posttranscriptional regulatory elements from the mammalian genome by selecting for rescue of virus replication. Viruses rescued by this method contained a novel element with homology to rodent intracisternal A-particle (IAP) retroelements. A functional element was contained within a 247-nucleotide fragment named RNA transport element (RTE), which was able to promote replication of the Rev- and RRE-defective HIV-1 in both human lymphoid cell lines and primary lymphocytes, demonstrating its potent posttranscriptional function. RTE was functional in many cell types, indicating that the cellular factors that recognize RTE are widely expressed and evolutionarily conserved. RTE also promoted RNA export fromXenopus oocyte nuclei. RTE-mediated RNA transport was CRM1 independent, and RTE did not show high affinity for binding to mRNA export factor TAP/NXF1. Since CRM1 and TAP/NXF1 are critical export receptors associated with the two recognized mRNA export pathways, these results suggest that RTE functions via a distinct export mechanism. Taken together, our results identify a novel posttranscriptional control element that uses a conserved cellular export mechanism.

scholarly journals Negative and Positive mRNA Splicing Elements Act Competitively To Regulate Human Immunodeficiency Virus Type 1 Vif Gene Expression

2008 ◽  
Vol 82 (8) ◽  
pp. 3921-3931 ◽  
Author(s):  
C. M. Exline ◽  
Z. Feng ◽  
C. M. Stoltzfus
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Regulatory Elements ◽  
Mrna Splicing ◽  
Viral Mrnas ◽  
Exon 2 ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNAs are produced by alternative splicing of the primary HIV-1 RNA transcripts. In addition, approximately half of the viral RNA remains unspliced and is used as genomic RNA and as mRNA for the Gag and Pol gene products. Regulation of splicing at the HIV-1 3′ splice sites (3′ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation occurs through the binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3′ss A1, which produces single-spliced vif mRNA and promotes the inclusion of HIV exon 2 into both completely and incompletely spliced viral mRNAs, is increased by optimizing the 5′ splice site (5′ss) downstream of exon 2 (5′ss D2). Here we show that the mutations within 5′ss D2 that are predicted to lower or increase the affinity of the 5′ss for U1 snRNP result in reduced or increased Vif expression, respectively. Splicing at 5′ss D2 was not necessary for the effect of 5′ss D2 on Vif expression. In addition, we have found that mutations of the GGGG motif proximal to the 5′ss D2 increase exon 2 inclusion and Vif expression. Finally, we report the presence of a novel exonic splicing enhancer (ESE) element within the 5′-proximal region of exon 2 that facilitates both exon inclusion and Vif expression. This ESE binds specifically to the cellular SR protein SRp75. Our results suggest that the 5′ss D2, the proximal GGGG silencer, and the ESE act competitively to determine the level of vif mRNA splicing and Vif expression. We propose that these positive and negative splicing elements act together to allow the accumulation of vif mRNA and unspliced HIV-1 mRNA, compatible with optimal virus replication.

scholarly journals RNA Trafficking Signals in Human Immunodeficiency Virus Type 1

2001 ◽  
Vol 21 (6) ◽  
pp. 2133-2143 ◽  
Author(s):  
Andrew J. Mouland ◽  
Hongbin Xu ◽  
Hongyi Cui ◽  
Winfried Krueger ◽  
Trent P. Munro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Rna Transport ◽  
Rna Trafficking ◽  
Trafficking Pathway ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Intracellular trafficking of retroviral RNAs is a potential mechanism to target viral gene expression to specific regions of infected cells. Here we show that the human immunodeficiency virus type 1 (HIV-1) genome contains two sequences similar to the hnRNP A2 response element (A2RE), a cis-acting RNA trafficking sequence that binds to the trans-acting trafficking factor, hnRNP A2, and mediates a specific RNA trafficking pathway characterized extensively in oligodendrocytes. The two HIV-1 sequences, designated A2RE-1, within the major homology region of the gag gene, and A2RE-2, in a region of overlap between the vpr andtat genes, both bind to hnRNP A2 in vitro and are necessary and sufficient for RNA transport in oligodendrocytes in vivo. A single base change (A8G) in either sequence reduces hnRNP A2 binding and, in the case of A2RE-2, inhibits RNA transport. A2RE-mediated RNA transport is microtubule and hnRNP A2 dependent. Differentially labelledgag and vpr RNAs, containing A2RE-1 and A2RE-2, respectively, coassemble into the same RNA trafficking granules and are cotransported to the periphery of the cell. tat RNA, although it contains A2RE-2, is not transported as efficiently asvpr RNA. An A2RE/hnRNP A2-mediated trafficking pathway for HIV RNA is proposed, and the role of RNA trafficking in targeting HIV gene expression is discussed.

scholarly journals Upstream stimulating factor affects human immunodeficiency virus type 1 (HIV-1) long terminal repeat-directed transcription in a cell-specific manner, independently of the HIV-1 subtype and the core-negative regulatory element

2001 ◽  
Vol 82 (3) ◽  
pp. 547-559 ◽  
Author(s):  
Mojgan H. Naghavi ◽  
Mario C. Estable ◽  
Stefan Schwartz ◽  
Robert G. Roeder ◽  
Anders Vahlne
Keyword(s):  
Human Immunodeficiency Virus ◽  
Regulatory Element ◽  
Negative Regulatory Element ◽  
The Core ◽  
Immunodeficiency Virus ◽  
Specific Manner ◽  
A Cell ◽  
Stimulating Factor ◽  
Hiv 1

Human immunodeficiency virus type 1 (HIV-1) is classified into subtypes on the basis of phylogenetic analysis of sequence differences. Inter- and intra-subtype polymorphism extends throughout the genome, including the long terminal repeat (LTR). In this study, the importance of the upstream stimulating factor (USF)-binding site (E-box) in the core-negative regulatory element (NRE) of the LTR of HIV-1 subtypes A, B, C, D, E and G was investigated. In vivo, USF was found to repress transcription directed from representative HIV-1 LTR sequences of all the subtypes tested in an epithelial cell line, yet activate the same transcription in a T-cell line. Mutation of the core-NRE USF site of the representative subtype B LTR did not affect the cell-specific, subtype-independent, dual role of USF. In vitro binding assays showed that recombinant USF43 interacts with the core-NRE from subtypes B and C, but not A, D, E or G. Thus, USF affects LTR-directed transcription in a cell-specific manner, independently of both the HIV-1 subtype from which the LTR was derived and the core-NRE USF site sequences.

scholarly journals Concerted Action of Multiple cis-Acting Sequences Is Required for Rev Dependence of Late Human Immunodeficiency Virus Type 1 Gene Expression

2000 ◽  
Vol 74 (22) ◽  
pp. 10822-10826 ◽  
Author(s):  
Marcus Graf ◽  
Alexandra Bojak ◽  
Ludwig Deml ◽  
Kurt Bieler ◽  
Hans Wolf ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Export ◽  
Regulatory Elements ◽  
Cis Acting ◽  
Immunodeficiency Virus ◽  
Responsive Element ◽  
Hiv 1

ABSTRACT Based on the human immunodeficiency virus type 1 (HIV-1)gag gene, subgenomic reporter constructs have been established allowing the contributions of differentcis-acting elements to the Rev dependency of late HIV-1 gene products to be determined. Modification of intragenic regulatory elements achieved by adapting the codon usage of the complete gene to highly expressed mammalian genes resulted in constitutive nuclear export allowing high levels of Gag expression independent from the Rev/Rev-responsive element system and irrespective of the absence or presence of the isolated major splice donor. Leptomycin B inhibitor studies revealed that the RNAs derived from the codon-optimizedgag gene lacking AU-rich inhibitory elements are directed to a distinct, CRM1-independent, nuclear export pathway.

scholarly journals Splicing Regulatory Elements within tat Exon 2 of Human Immunodeficiency Virus Type 1 (HIV-1) Are Characteristic of Group M but Not Group O HIV-1 Strains

1999 ◽  
Vol 73 (12) ◽  
pp. 9764-9772 ◽  
Author(s):  
Patricia S. Bilodeau ◽  
Jeffrey K. Domsic ◽  
C. Martin Stoltzfus
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Splice Site ◽  
Regulatory Elements ◽  
Splice Sites ◽  
Exon 2 ◽  
Immunodeficiency Virus ◽  
Splicing Regulatory Elements ◽  
Hiv 1

ABSTRACT In the NL4-3 strain of human immunodeficiency virus type 1 (HIV-1), regulatory elements responsible for the relative efficiencies of alternative splicing at the tat, rev, and theenv/nef 3′ splice sites (A3 through A5) are contained within the region of tat exon 2 and its flanking sequences. Two elements affecting splicing of tat, rev, and env/nef mRNAs have been localized to this region. First, an exon splicing silencer (ESS2) in NL4-3, located approximately 70 nucleotides downstream from the 3′ splice site used to generatetat mRNA, acts specifically to inhibit splicing at this splice site. Second, the A4b 3′ splice site, which is the most downstream of the three rev 3′ splice sites, also serves as an element inhibiting splicing at the env/nef 3′ splice site A5. These elements are conserved in some but not all HIV-1 strains, and the effects of these sequence changes on splicing have been investigated in cell transfection and in vitro splicing assays. SF2, another clade B virus and member of the major (group M) viruses, has several sequence changes within ESS2 and uses a differentrev 3′ splice site. However, splicing is inhibited by the two elements similarly to NL4-3. As with the NL4-3 strain, the SF2 A4b AG dinucleotide overlaps an A5 branchpoint, and thus the inhibitory effect may result from competition of the same site for two different splicing factors. The sequence changes in ANT70C, a member of the highly divergent outlier (group O) viruses, are more extensive, and ESS2 activity in tat exon 2 is not present. Group O viruses also lack the rev 3′ splice site A4b, which is conserved in all group M viruses. Mutagenesis of the most downstream rev3′ splice site of ANT70C does not increase splicing at A5, and all of the branchpoints are upstream of the two rev 3′ splice sites. Thus, splicing regulatory elements in tat exon 2 which are characteristic of most group M HIV-1 strains are not present in group O HIV-1 strains.

scholarly journals Interaction of YB-1 with human immunodeficiency virus type 1 Tat and TAR RNA modulates viral promoter activity

1999 ◽  
Vol 80 (10) ◽  
pp. 2629-2638 ◽  
Author(s):  
Sameer A. Ansari ◽  
Mahmut Safak ◽  
Gary L. Gallia ◽  
Bassel E. Sawaya ◽  
Shohreh Amini ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Rna Binding ◽  
Regulatory Element ◽  
Binding Activity ◽  
Full Length ◽  
Immunodeficiency Virus ◽  
Hiv 1

Transcriptional regulation of the human immunodeficiency virus type 1 (HIV-1) genome is mediated by viral and cellular factors. TAR, an unusual RNA regulatory element with a stem–bulge–loop structure at the 5′ ends of all nascent viral transcripts is critical for HIV-1 transcription. TAR is the target for Tat, a viral transcription factor encoded early in the HIV-1 life-cycle and essential for gene expression. Evidence demonstrating the interaction of a cellular ssDNA/RNA binding protein, YB-1, with TAR through a region which is important for Tat interaction is presented. Interestingly, results from protein–protein interaction studies revealed that YB-1 can also form a complex with Tat. Results from mapping experiments suggest that while the region spanning aa 125–203 within YB-1 is essential for its association with TAR, a truncated YB-1 spanning aa 1–125 can weakly bind to Tat. Functionally, overexpression of full-length YB-1 enhanced Tat-induced activation of the HIV-1 minimal promoter containing TAR sequences, whereas mutant YB-1 with no ability to bind to Tat and TAR failed to affect Tat-mediated activation. Expression of mutant YB-1(1–125), which binds to Tat but not RNA, decreased Tat- mediated enhancement of virus transcription. These observations suggest that while full-length YB-1 may function as a facilitator and, by interaction with both Tat and TAR, increase the level of Tat:TAR association, mutant YB-1 with no TAR binding activity, by complexing with Tat, may prevent Tat interaction with TAR. The importance of these findings in light of the proposed mechanism of Tat function is discussed.

scholarly journals The Secondary Structure of the Human Immunodeficiency Virus Type 1 Transcript Modulates Viral Splicing and Infectivity

2008 ◽  
Vol 82 (16) ◽  
pp. 8038-8050 ◽  
Author(s):  
Joseph A. Jablonski ◽  
Emanuele Buratti ◽  
Cristiana Stuani ◽  
Massimo Caputi
Keyword(s):  
Human Immunodeficiency Virus ◽  
Secondary Structure ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Regulatory Element ◽  
Spatial Location ◽  
Splicing Factors ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Splicing of human immunodeficiency virus type 1 (HIV-1) exon 6D is regulated by the presence of a complex splicing regulatory element (SRE) sequence that interacts with the splicing factors hnRNP H and SC35. In this work, we show that, in the context of the wild-type viral sequence, hnRNP H acts as a repressor of exon 6D inclusion independent of its binding to the SRE. However, hnRNP H binding to the SRE acts as an enhancer of exon 6D inclusion in the presence of a critical T-to-C mutation. These seemingly contrasting functional properties of hnRNP H appear to be caused by a change in the RNA secondary structure induced by the T-to-C mutation that affects the spatial location of bound hnRNP H with respect to the exon 6D splicing determinants. We propose a new regulatory mechanism mediated by RNA folding that may also explain the dual properties of hnRNP H in splicing regulation.

scholarly journals Presence of negative and positive cis-acting RNA splicing elements within and flanking the first tat coding exon of human immunodeficiency virus type 1.

1994 ◽  
Vol 14 (6) ◽  
pp. 3960-3970 ◽  
Author(s):  
B A Amendt ◽  
D Hesslein ◽  
L J Chang ◽  
C M Stoltzfus
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Splice Site ◽  
Rna Splicing ◽  
Regulatory Element ◽  
Cis Acting ◽  
Immunodeficiency Virus ◽  
Hiv 1

The human immunodeficiency virus type 1 (HIV-1) RNA follows a complex splicing pathway in which a single primary transcript either remains unspliced or is alternatively spliced to more than 30 different singly and multiply spliced mRNAs. We have used an in vitro splicing assay to identify cis elements within the viral genome that regulate HIV-1 RNA splicing. A novel splicing regulatory element (SRE) within the first tat coding exon has been detected. This element specifically inhibits splicing at the upstream 3' splice site flanking this tat exon. The element only functions when in the sense orientation and is position dependent when inserted downstream of a heterologous 3' splice site. In vivo, an HIV-1 SRE mutant demonstrated a decrease in unspliced viral RNA, increased levels of single- and double-spliced tat mRNA, and reduced levels of env and rev mRNAs. In addition to the negative cis-acting SRE, the flanking 5' splice site downstream of the first tat coding exon acts positively to increase splicing at the upstream 3' splice sites. These results are consistent with hypotheses of bridging interactions between cellular factors that bind to the 5' splice site and those that bind at the upstream 3' splice site.

scholarly journals PSF Acts through the Human Immunodeficiency Virus Type 1 mRNA Instability Elements To Regulate Virus Expression

2003 ◽  
Vol 23 (18) ◽  
pp. 6618-6630 ◽  
Author(s):  
Andrei S. Zolotukhin ◽  
Daniel Michalowski ◽  
Jenifer Bear ◽  
Sergey V. Smulevitch ◽  
Abdulmaged M. Traish ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Export ◽  
Regulatory Elements ◽  
Nucleocytoplasmic Transport ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV) gag/pol and env mRNAs contain cis-acting regulatory elements (INS) that impair stability, nucleocytoplasmic transport, and translation by unknown mechanisms. This downregulation can be counteracted by the viral Rev protein, resulting in efficient export and expression of these mRNAs. Here, we show that the INS region in HIV-1 gag mRNA is a high-affinity ligand of p54nrb/PSF, a heterodimeric transcription/splicing factor. Both subunits bound INS RNA in vitro with similar affinity and specificity. Using an INS-containing subgenomic gag mRNA, we show that it specifically associated with p54nrb in vivo and that PSF inhibited its expression, acting via INS. Studying the authentic HIV-1 mRNAs produced from an infectious molecular clone, we found that PSF affected specifically the INS-containing, Rev-dependent transcripts encoding Gag-Pol and Env. Both subunits contained nuclear export and nuclear retention signals, whereas p54nrb was continuously exported from the nucleus and associated with INS-containing mRNA in the cytoplasm, suggesting its additional role at late steps of mRNA metabolism. Thus, p54nrb and PSF have properties of key factors mediating INS function and likely define a novel mRNA regulatory pathway that is hijacked by HIV-1.

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