Involvement of long terminal repeat U3 sequences overlapping the transcription control region in human immunodeficiency virus type 1 mRNA 3' end formation

In retroviral proviruses, the poly(A) site is present in both long terminal repeats (LTRs) but used only in the 3' position. One mechanism to account for this selective poly(A) site usage is that LTR U3 sequences, transcribed only from the 3' poly(A) site, are required in the RNA for efficient processing. To test this possibility, mutations were made in the human immunodeficiency virus type 1 (HIV-1) U3 region and the mutated LTRs were inserted into simple and complex transcription units. HIV-1 poly(A) site usage was then quantitated by S1 nuclease analysis following transfection of each construct into human 293 cells. The results showed that U3 sequences confined to the transcription control region were required for efficient usage of the HIV-1 poly(A) site, even when it was placed 1.5 kb from the promoter. Although the roles of U3 in processing and transcription activation were separable, optimal 3' end formation was partly dependent on HIV-1 enhancer and SP1 binding site sequences.

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Relative roles of signals upstream of AAUAAA and promoter proximity in regulation of human immunodeficiency virus type 1 mRNA 3' end formation.

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5555 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5555-5562 ◽

Cited By ~ 15

Author(s):

J D DeZazzo ◽

J M Scott ◽

M J Imperiale

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Dependent Manner ◽

Immunodeficiency Virus ◽

A Site ◽

The Impact ◽

Hiv 1 ◽

Rna Levels

At least two mechanisms have been implicated in regulating poly(A) site use in human immunodeficiency virus type 1 (HIV-1): inhibition of basal signals within 500 nucleotides (nt) of the cap site, leading to specific suppression of the 5' poly(A) site, and stimulation of basal signals by long terminal repeat U3 sequences, leading to specific activation of the 3' poly(A) site. We determined the relative contributions of these mechanisms in a HeLa cell transcription/processing reaction and by transient transfection analysis. In vitro, the efficiency of basal signals is equivalent close to (270 nt) and far from (1,080 nt) the promoter and is stimulated at least 30-fold in both positions by upstream U3 sequences. In vivo, U3 sequences also enhance processing at both positions. There are two additional effects when the poly(A) site is close to the cap site: at least a 15-fold reduction in total RNA levels and a 5-fold decrease in relative levels of RNA processed at the HIV-1 site in constructs containing U3. Both effects are overcome by insertion of upstream splicing signals in an orientation-dependent manner. Splicing appears to influence poly(A)+ RNA levels by two distinct mechanisms: stabilizing nuclear transcripts and directly stimulating 3' end formation. It is proposed that upstream elements play major roles in regulating poly(A) site choice and in controlling the subsequent fate of polyadenylated RNA. The impact of these findings on mechanisms of mRNA biogenesis in the HIV-1 provirus is discussed.

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Relative roles of signals upstream of AAUAAA and promoter proximity in regulation of human immunodeficiency virus type 1 mRNA 3' end formation

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5555-5562.1992 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5555-5562

Author(s):

J D DeZazzo ◽

J M Scott ◽

M J Imperiale

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Dependent Manner ◽

Immunodeficiency Virus ◽

A Site ◽

The Impact ◽

Hiv 1 ◽

Rna Levels

At least two mechanisms have been implicated in regulating poly(A) site use in human immunodeficiency virus type 1 (HIV-1): inhibition of basal signals within 500 nucleotides (nt) of the cap site, leading to specific suppression of the 5' poly(A) site, and stimulation of basal signals by long terminal repeat U3 sequences, leading to specific activation of the 3' poly(A) site. We determined the relative contributions of these mechanisms in a HeLa cell transcription/processing reaction and by transient transfection analysis. In vitro, the efficiency of basal signals is equivalent close to (270 nt) and far from (1,080 nt) the promoter and is stimulated at least 30-fold in both positions by upstream U3 sequences. In vivo, U3 sequences also enhance processing at both positions. There are two additional effects when the poly(A) site is close to the cap site: at least a 15-fold reduction in total RNA levels and a 5-fold decrease in relative levels of RNA processed at the HIV-1 site in constructs containing U3. Both effects are overcome by insertion of upstream splicing signals in an orientation-dependent manner. Splicing appears to influence poly(A)+ RNA levels by two distinct mechanisms: stabilizing nuclear transcripts and directly stimulating 3' end formation. It is proposed that upstream elements play major roles in regulating poly(A) site choice and in controlling the subsequent fate of polyadenylated RNA. The impact of these findings on mechanisms of mRNA biogenesis in the HIV-1 provirus is discussed.

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Mapping Protease Inhibitor Resistance to Human Immunodeficiency Virus Type 1 Sequence Polymorphisms within Patients

Journal of Virology ◽

10.1128/jvi.01570-07 ◽

2007 ◽

Vol 81 (24) ◽

pp. 13598-13607 ◽

Cited By ~ 16

Author(s):

Art F. Y. Poon ◽

Sergei L. Kosakovsky Pond ◽

Douglas D. Richman ◽

Simon D. W. Frost

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Specific Marker ◽

Naive Patient ◽

Immunodeficiency Virus ◽

A Site ◽

Inhibitor Resistance ◽

Hiv 1

ABSTRACT Resistance genotyping provides an important resource for the clinical management of patients infected with human immunodeficiency virus type 1 (HIV-1). However, resistance to protease (PR) inhibitors (PIs) is a complex phenotype shaped by interactions among nearly half of the residues in HIV-1 PR. Previous studies of the genetic basis of PI resistance focused on fixed substitutions among populations of HIV-1, i.e., host-specific adaptations. Consequently, they are susceptible to a high false discovery rate due to founder effects. Here, we employ sequencing “mixtures” (i.e., ambiguous base calls) as a site-specific marker of genetic variation within patients that is independent of the phylogeny. We demonstrate that the transient response to selection by PIs is manifested as an excess of nonsynonymous mixtures. Using a sample of 5,651 PR sequences isolated from both PI-naive and -treated patients, we analyze the joint distribution of mixtures and eight PIs as a Bayesian network, which distinguishes residue-residue interactions from direct associations with PIs. We find that selection for resistance is associated with the emergence of nonsynonymous mixtures in two distinct groups of codon sites clustered along the substrate cleft and distal regions of PR, respectively. Within-patient evolution at several positions is independent of PIs, including those formerly postulated to be involved in resistance. These positions are under strong positive selection in the PI-naive patient population, implying that other factors can produce spurious associations with resistance, e.g., mutational escape from the immune response.

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