scholarly journals Function of a Bovine Papillomavirus Type 1 Exonic Splicing Suppressor Requires a Suboptimal Upstream 3′ Splice Site

1999 ◽  
Vol 73 (1) ◽  
pp. 29-36 ◽  
Author(s):  
Zhi Ming Zheng ◽  
Pei-jun He ◽  
Carl C. Baker
Keyword(s):  
Splice Site ◽  
Bovine Papillomavirus ◽  
Rich Region ◽  
Rous Sarcoma ◽  
Virus Life Cycle ◽  
Immunodeficiency Virus ◽  
Sarcoma Virus ◽  
In Vitro Splicing

ABSTRACT Alternative splicing is an important mechanism for the regulation of bovine papillomavirus type 1 (BPV-1) gene expression during the virus life cycle. Previous studies in our laboratory have identified two purine-rich exonic splicing enhancers (ESEs), SE1 and SE2, located between two alternative 3′ splice sites at nucleotide (nt) 3225 and nt 3605. Further analysis of BPV-1 late-pre-mRNA splicing in vitro revealed a 48-nt pyrimidine-rich region immediately downstream of SE1 that inhibits utilization of the nt 3225 3′ splice site. This inhibitory element, which we named an exonic splicing suppressor (ESS), has a U-rich 5′ end, a C-rich central part, and an AG-rich 3′ end (Z. M. Zheng, P. He, and C. C. Baker, J. Virol. 70:4691–4699, 1996). The present study utilized in vitro splicing of both homologous and heterologous pre-mRNAs to further characterize the ESS. The BPV-1 ESS was inserted downstream of the 3′ splice site in the BPV-1 late pre-mRNA, Rous sarcoma virussrc pre-mRNA, human immunodeficiency virustat-rev pre-mRNA, and Drosophila dsx pre-mRNA, all containing a suboptimal 3′ splice site, and in the human β-globin pre-mRNA, which contains a constitutive 3′ splice site. These studies demonstrated that suppression of splicing by the BPV-1 ESS requires an upstream suboptimal 3′ splice site but not an upstream ESE. Furthermore, the ESS functions when located either upstream or downstream of BPV-1 SE1. Mutational analyses demonstrated that the function of the ESS is sequence dependent and that only the C-rich region of the ESS is essential for suppression of splicing in all the pre-mRNAs tested.

scholarly journals Optimization of a Weak 3′ Splice Site Counteracts the Function of a Bovine Papillomavirus Type 1 Exonic Splicing Suppressor In Vitro and In Vivo

2000 ◽  
Vol 74 (13) ◽  
pp. 5902-5910 ◽  
Author(s):  
Zhi-Ming Zheng ◽  
Jesse Quintero ◽  
Eric S. Reid ◽  
Christian Gocke ◽  
Carl C. Baker
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Splicing Factors ◽  
Bovine Papillomavirus ◽  
Splicing Pattern ◽  
In Vitro Splicing

ABSTRACT Alternative splicing is a critical component of the early to late switch in papillomavirus gene expression. In bovine papillomavirus type 1 (BPV-1), a switch in 3′ splice site utilization from an early 3′ splice site at nucleotide (nt) 3225 to a late-specific 3′ splice site at nt 3605 is essential for expression of the major capsid (L1) mRNA. Three viral splicing elements have recently been identified between the two alternative 3′ splice sites and have been shown to play an important role in this regulation. A bipartite element lies approximately 30 nt downstream of the nt 3225 3′ splice site and consists of an exonic splicing enhancer (ESE), SE1, followed immediately by a pyrimidine-rich exonic splicing suppressor (ESS). A second ESE (SE2) is located approximately 125 nt downstream of the ESS. We have previously demonstrated that the ESS inhibits use of the suboptimal nt 3225 3′ splice site in vitro through binding of cellular splicing factors. However, these in vitro studies did not address the role of the ESS in the regulation of alternative splicing. In the present study, we have analyzed the role of the ESS in the alternative splicing of a BPV-1 late pre-mRNA in vivo. Mutation or deletion of just the ESS did not significantly change the normal splicing pattern where the nt 3225 3′ splice site is already used predominantly. However, a pre-mRNA containing mutations in SE2 is spliced predominantly using the nt 3605 3′ splice site. In this context, mutation of the ESS restored preferential use of the nt 3225 3′ splice site, indicating that the ESS also functions as a splicing suppressor in vivo. Moreover, optimization of the suboptimal nt 3225 3′ splice site counteracted the in vivo function of the ESS and led to preferential selection of the nt 3225 3′ splice site even in pre-mRNAs with SE2 mutations. In vitro splicing assays also showed that the ESS is unable to suppress splicing of a pre-mRNA with an optimized nt 3225 3′ splice site. These data confirm that the function of the ESS requires a suboptimal upstream 3′ splice site. A surprising finding of our study is the observation that SE1 can stimulate both the first and the second steps of splicing.

scholarly journals Mechanistic Differences between Nucleic Acid Chaperone Activities of the Gag Proteins of Rous Sarcoma Virus and Human Immunodeficiency Virus Type 1 Are Attributed to the MA Domain

2014 ◽  
Vol 88 (14) ◽  
pp. 7852-7861 ◽  
Author(s):  
Tiffiny D. Rye-McCurdy ◽  
Shorena Nadaraia-Hoke ◽  
Nicole Gudleski-O'Regan ◽  
John M. Flanagan ◽  
Leslie J. Parent ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Inositol Phosphates ◽  
Chaperone Activity ◽  
Chaperone Function ◽  
Rous Sarcoma ◽  
Immunodeficiency Virus ◽  
Sarcoma Virus ◽  
Hiv 1

ABSTRACTHost cell tRNAs are recruited for use as primers to initiate reverse transcription in retroviruses. Human immunodeficiency virus type 1 (HIV-1) uses tRNALys3as the replication primer, whereas Rous sarcoma virus (RSV) uses tRNATrp. The nucleic acid (NA) chaperone function of the nucleocapsid (NC) domain of HIV-1 Gag is responsible for annealing tRNALys3to the genomic RNA (gRNA) primer binding site (PBS). Compared to HIV-1, little is known about the chaperone activity of RSV Gag. In this work, using purified RSV Gag containing an N-terminal His tag and a deletion of the majority of the protease domain (H6.Gag.3h), gel shift assays were used to monitor the annealing of tRNATrpto a PBS-containing RSV RNA. Here, we show that similar to HIV-1 Gag lacking the p6 domain (GagΔp6), RSV H6.Gag.3h is a more efficient chaperone on a molar basis than NC; however, in contrast to the HIV-1 system, both RSV H6.Gag.3h and NC have comparable annealing rates at protein saturation. The NC domain of RSV H6.Gag.3h is required for annealing, whereas deletion of the matrix (MA) domain, which stimulates the rate of HIV-1 GagΔp6 annealing, has little effect on RSV H6.Gag.3h chaperone function. Competition assays confirmed that RSV MA binds inositol phosphates (IPs), but in contrast to HIV-1 GagΔp6, IPs do not stimulate RSV H6.Gag.3h chaperone activity unless the MA domain is replaced with HIV-1 MA. We conclude that differences in the MA domains are primarily responsible for mechanistic differences in RSV and HIV-1 Gag NA chaperone function.IMPORTANCEMounting evidence suggests that the Gag polyprotein is responsible for annealing primer tRNAs to the PBS to initiate reverse transcription in retroviruses, but only HIV-1 Gag chaperone activity has been demonstratedin vitro. Understanding RSV Gag's NA chaperone function will allow us to determine whether there is a common mechanism among retroviruses. This report shows for the first time that full-length RSV Gag lacking the protease domain is a highly efficient NA chaperonein vitro, and NC is required for this activity. In contrast to results obtained for HIV-1 Gag, due to the weak nucleic acid binding affinity of the RSV MA domain, inositol phosphates do not regulate RSV Gag-facilitated tRNA annealing despite the fact that they bind to MA. These studies provide insight into the viral regulation of tRNA primer annealing, which is a potential target for antiretroviral therapy.

scholarly journals Utilization of the Bovine Papillomavirus Type 1 Late-Stage-Specific Nucleotide 3605 3′ Splice Site Is Modulated by a Novel Exonic Bipartite Regulator but Not by an Intronic Purine-Rich Element

2000 ◽  
Vol 74 (22) ◽  
pp. 10612-10622 ◽  
Author(s):  
Zhi-Ming Zheng ◽  
Eric S. Reid ◽  
Carl C. Baker
Keyword(s):  
Life Cycle ◽  
Splice Site ◽  
Late Stage ◽  
Keratinocyte Differentiation ◽  
Bovine Papillomavirus ◽  
Late Gene Expression ◽  
Virus Life Cycle ◽  
Branch Point Sequence

ABSTRACT Bovine papillomavirus type 1 (BPV-1) late gene expression is regulated at both transcriptional and posttranscriptional levels. Maturation of the capsid protein (L1) pre-mRNA requires a switch in 3′ splice site utilization. This switch involves activation of the nucleotide (nt) 3605 3′ splice site, which is utilized only in fully differentiated keratinocytes during late stages of the virus life cycle. Our previous studies of the mechanisms that regulate BPV-1 alternative splicing identified three cis-acting elements between these two splice sites. Two purine-rich exonic splicing enhancers, SE1 and SE2, are essential for preferential utilization of the nt 3225 3′ splice site at early stages of the virus life cycle. Another cis-acting element, exonic splicing suppressor 1 (ESS1), represses use of the nt 3225 3′ splice site. In the present study, we investigated the late-stage-specific nt 3605 3′ splice site and showed that it has suboptimal features characterized by a nonconsensus branch point sequence and a weak polypyrimidine track with interspersed purines. In vitro and in vivo experiments showed that utilization of the nt 3605 3′ splice site was not affected by SE2, which is intronically located with respect to the nt 3605 3′ splice site. The intronic location and sequence composition of SE2 are similar to those of the adenovirus IIIa repressor element, which has been shown to inhibit use of a downstream 3′ splice site. Further studies demonstrated that the nt 3605 3′ splice site is controlled by a novel exonic bipartite element consisting of an AC-rich exonic splicing enhancer (SE4) and an exonic splicing suppressor (ESS2) with a UGGU motif. Functionally, this newly identified bipartite element resembles the bipartite element composed of SE1 and ESS1. SE4 also functions on a heterologous 3′ splice site. In contrast, ESS2 functions as an exonic splicing suppressor only in a 3′-splice-site-specific and enhancer-specific manner. Our data indicate that BPV-1 splicing regulation is very complex and is likely to be controlled by multiple splicing factors during keratinocyte differentiation.

scholarly journals Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases.

1992 ◽  
Vol 12 (5) ◽  
pp. 2331-2338 ◽  
Author(s):  
J Kulkosky ◽  
K S Jones ◽  
R A Katz ◽  
J P Mack ◽  
A M Skalka
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Rous Sarcoma Virus ◽  
Amino Acid Sequences ◽  
Rous Sarcoma ◽  
Essential Components ◽  
Immunodeficiency Virus ◽  
Sarcoma Virus ◽  
E Region

Our comparison of deduced amino acid sequences for retroviral/retrotransposon integrase (IN) proteins of several organisms, including Drosophila melanogaster and Schizosaccharomyces pombe, reveals strong conservation of a constellation of amino acids characterized by two invariant aspartate (D) residues and a glutamate (E) residue, which we refer to as the D,D(35)E region. The same constellation is found in the transposases of a number of bacterial insertion sequences. The conservation of this region suggests that the component residues are involved in DNA recognition, cutting, and joining, since these properties are shared among these proteins of divergent origin. We introduced amino acid substitutions in invariant residues and selected conserved and nonconserved residues throughout the D,D(35)E region of Rous sarcoma virus IN and in human immunodeficiency virus IN and assessed their effect upon the activities of the purified, mutant proteins in vitro. Changes of the invariant and conserved residues typically produce similar impairment of both viral long terminal repeat (LTR) oligonucleotide cleavage referred to as the processing reaction and the subsequent joining of the processed LTR-based oligonucleotides to DNA targets. The severity of the defects depended upon the site and the nature of the amino acid substitution(s). All substitutions of the invariant acidic D and E residues in both Rous sarcoma virus and human immunodeficiency virus IN dramatically reduced LTR oligonucleotide processing and joining to a few percent or less of wild type, suggesting that they are essential components of the active site for both reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The 5′ and 3′ TAR Elements of Human Immunodeficiency Virus Exert Effects at Several Points in the Virus Life Cycle

1998 ◽  
Vol 72 (11) ◽  
pp. 9217-9223 ◽  
Author(s):  
Atze T. Das ◽  
Bep Klaver ◽  
Ben Berkhout
Keyword(s):  
Human Immunodeficiency Virus ◽  
Life Cycle ◽  
Reverse Transcription ◽  
Wild Type ◽  
Rna Packaging ◽  
Virus Life Cycle ◽  
Activation Of Transcription ◽  
Immunodeficiency Virus

ABSTRACT The human immunodeficiency virus type 1 RNA genome contains a terminal repeat (R) sequence that encodes the TAR hairpin motif, which has been implicated in Tat-mediated activation of transcription. More recently, a variety of other functions have been proposed for this structured RNA element. To determine the replicative roles of the 5′ and 3′ TAR hairpins, we analyzed multiple steps in the life cycle of wild-type and mutant viruses. A structure-destabilizing mutation was introduced in either the 5′, the 3′, or both TAR motifs of the proviral genome. As expected, opening of the 5′ TAR hairpin caused a transcription defect. Because the level of protein expression was not similarly reduced, the translation of this mRNA was improved. No effect of the 3′ hairpin on transcription and translation was measured. Mutations of the 5′ and 3′ hairpin structures reduced the efficiency of RNA packaging to similar extents, and RNA packaging was further reduced in the 5′ and 3′ TAR double mutant. Upon infection of cells with these virions, a reduced amount of reverse transcription products was synthesized by the TAR mutant. However, no net reverse transcription defect was observed after correction for the reduced level of virion RNA. This result was confirmed in in vitro reverse transcription assays. These data indicate that the 5′ and 3′ TAR motifs play important roles in several steps of the replication cycle, but these structures have no significant effect on the mechanism of reverse transcription.

Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases

1992 ◽  
Vol 12 (5) ◽  
pp. 2331-2338
Author(s):  
J Kulkosky ◽  
K S Jones ◽  
R A Katz ◽  
J P Mack ◽  
A M Skalka
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Rous Sarcoma Virus ◽  
Amino Acid Sequences ◽  
Rous Sarcoma ◽  
Essential Components ◽  
Immunodeficiency Virus ◽  
Sarcoma Virus ◽  
E Region

Our comparison of deduced amino acid sequences for retroviral/retrotransposon integrase (IN) proteins of several organisms, including Drosophila melanogaster and Schizosaccharomyces pombe, reveals strong conservation of a constellation of amino acids characterized by two invariant aspartate (D) residues and a glutamate (E) residue, which we refer to as the D,D(35)E region. The same constellation is found in the transposases of a number of bacterial insertion sequences. The conservation of this region suggests that the component residues are involved in DNA recognition, cutting, and joining, since these properties are shared among these proteins of divergent origin. We introduced amino acid substitutions in invariant residues and selected conserved and nonconserved residues throughout the D,D(35)E region of Rous sarcoma virus IN and in human immunodeficiency virus IN and assessed their effect upon the activities of the purified, mutant proteins in vitro. Changes of the invariant and conserved residues typically produce similar impairment of both viral long terminal repeat (LTR) oligonucleotide cleavage referred to as the processing reaction and the subsequent joining of the processed LTR-based oligonucleotides to DNA targets. The severity of the defects depended upon the site and the nature of the amino acid substitution(s). All substitutions of the invariant acidic D and E residues in both Rous sarcoma virus and human immunodeficiency virus IN dramatically reduced LTR oligonucleotide processing and joining to a few percent or less of wild type, suggesting that they are essential components of the active site for both reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals PR Domain of Rous Sarcoma Virus Gag Causes an Assembly/Budding Defect in Insect Cells

2001 ◽  
Vol 75 (9) ◽  
pp. 4407-4412 ◽  
Author(s):  
Marc C. Johnson ◽  
Heather M. Scobie ◽  
Volker M. Vogt
Keyword(s):  
Rous Sarcoma Virus ◽  
Gag Protein ◽  
Gag Proteins ◽  
Rous Sarcoma ◽  
Immunodeficiency Virus ◽  
Electron Microscopy Analysis ◽  
Sarcoma Virus ◽  
Microscopy Analysis ◽  
Pr Domain

ABSTRACT While baculovirus expression of Gag proteins from numerous retroviruses has led reliably to production of virus-like particles (VLPs), we observed that expression of Rous sarcoma virus Gag failed to produce VLPs. Transmission and scanning electron microscopy analysis revealed that the Gag protein reached the plasma membrane but was unable to correctly form particles. Addition of a myristylation signal had no effect on the budding defect, but deletion of the PR domain of Gag restored normal budding. The resulting VLPs were morphologically distinct from human immunodeficiency virus type 1 VLPs expressed in parallel.

scholarly journals Retrovirus-Specific Packaging of Aminoacyl-tRNA Synthetases with Cognate Primer tRNAs

2002 ◽  
Vol 76 (24) ◽  
pp. 13111-13115 ◽  
Author(s):  
Shan Cen ◽  
Hassan Javanbakht ◽  
Sunghoon Kim ◽  
Kiyotaka Shiba ◽  
Rebecca Craven ◽  
...  
Keyword(s):  
Leukemia Virus ◽  
Trna Synthetase ◽  
Moloney Murine Leukemia Virus ◽  
Trna Synthetases ◽  
Rous Sarcoma ◽  
Aminoacyl Trna Synthetases ◽  
Immunodeficiency Virus ◽  
Sarcoma Virus ◽  
Hiv 1

ABSTRACT The tRNAs used to prime reverse transcription in human immunodeficiency virus type 1 (HIV-1), Rous sarcoma virus (RSV), and Moloney murine leukemia virus (Mo-MuLV) are \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} , tRNATrp, and tRNAPro, respectively. Using antibodies to the three cognate human aminoacyl-tRNA synthetases, we found that only lysyl-tRNA synthetase (LysRS) is present in HIV-1, only tryptophanyl-tRNA synthetase (TrpRS) is present in RSV, and neither these two synthetases nor prolyl-tRNA synthetase (ProRS) is present in Mo-MuLV. LysRS and TrpRS are present in HIV-1 and RSV at approximately 25 and 12 molecules/virion, respectively. These results support the hypothesis that, in HIV-1 and RSV, the cognate aminoacyl-tRNA synthetase may be used as the signal for targeting the selective packaging of primer tRNAs into retroviruses. The absence of ProRS in Mo-MuLV is consistent with reports that selective packaging of tRNAPro in this virus is less important for achieving optimum annealing of the primer to Mo-MuLV genomic RNA.

Sign in / Sign up

Export Citation Format

Share Document