Marie-Louise Hammarskjold | University of Virginia (original) (raw)

Papers by Marie-Louise Hammarskjold

Research paper thumbnail of Identification of a cis-acting element in human immunodeficiency virus type 2 (HIV-2) that is responsive to the HIV-1 rev and human T-cell leukemia virus types I and II rex proteins

Journal of Virology, 1990

A simian virus 40 late replacement vector encoding human immunodeficiency virus type 1 (HIV-1) gp... more A simian virus 40 late replacement vector encoding human immunodeficiency virus type 1 (HIV-1) gp120 (pGP120) was used to define a region within the HIV-2 genome that could work as a rev-responsive element (RRE). Our previous work showed that gp120 expression in this system required a functional RRE in cis and required the rev protein in trans (M.-L. Hammarskjöld, J. Heimer, B. Hammarskjöld, I. Sangwan, L. Albert, and D. Rekosh, J. Virol. 63:1959-1966, 1989). Using pGP120, we first mapped an RRE to a 1,042-base-pair (bp) Sau3a fragment in the env region of HIV-2. Both HIV-1 rev (rev1) and HIV-2 rev (rev2) could work in conjunction with this fragment. Further mapping showed that a 272-bp subfragment within the 1,042-bp region was sufficient as an RRE. Surprisingly, the smaller fragment worked only with the rev1 protein and not with its homologous rev2 protein. In addition, the rev2 protein failed to function together with the RRE from HIV-1. We also utilized this system to examine th...

Research paper thumbnail of RNA and lessons from viruses

RNA (New York, N.Y.), 2015

Research paper thumbnail of The effect of viral regulatory protein expression on gene delivery by human immunodeficiency virus type 1 vectors produced in stable packaging cell lines

Journal of virology, 1997

We describe the generation of stable human immunodeficiency virus type 1 (HIV-1)-packaging lines ... more We describe the generation of stable human immunodeficiency virus type 1 (HIV-1)-packaging lines that constitutively express high levels of HIV-1 structural proteins in either a Rev-dependent or a Rev-independent fashion. These cell lines were used to assess gene transfer by using an HIV-1 vector expressing the hygromycin B resistance gene and to study the effects of Rev, Tat, and Nef on the vector titer. The Rev-independent cell lines were created by using gag-pol and env expression vectors that contain the Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE). Vector titers approaching 10(4) CFU/ml were routinely obtained with these cell lines, as well as with the Rev-dependent cell lines, with HeLa-CD4 cells as targets. The presence of Nef and Tat in the producer cell each increased the vector titer 5- to 10-fold. Rev, on the other hand, was absolutely essential for gene transfer, unless the MPMV CTE was present in the vector. In that case, by using the Rev-indepe...

Research paper thumbnail of Human immunodeficiency virus type 1 Pr55gag and Pr160gag-pol expressed from a simian virus 40 late replacement vector are efficiently processed and assembled into viruslike particles

Journal of Virology, 1990

Human immunodeficiency virus type 1 (HIV-1) gag and pol genes were expressed by using fragments o... more Human immunodeficiency virus type 1 (HIV-1) gag and pol genes were expressed by using fragments of the BH10 clone of HIV inserted into a simian virus 40 late replacement vector. An initial construct containing the entire coding regions of gag, pol, and vif produced only minute amounts of the gag precursor, Pr55gag. However, high-level expression was obtained when an additional sequence from the env gene (the rev-responsive element) was inserted 3' of vif in the correct orientation, and rev was provided in trans from a second vector. Western immunoblot analysis of transfected cells showed the presence of large amounts of both Pr55gag and Pr160gag-pol as well as all of the expected cleavage products. Electron microscopy of thin sections of transfected cells showed a multitude of viruslike particles. Both immature particles in the process of budding and particles containing the condensed core characteristic of HIV were observed. Analysis of the released viruslike particles showed t...

Research paper thumbnail of Human immunodeficiency virus env expression becomes Rev-independent if the env region is not defined as an intron

Journal of Virology, 1994

The human immunodeficiency virus (HIV) Rev protein functions to facilitate export of intron-conta... more The human immunodeficiency virus (HIV) Rev protein functions to facilitate export of intron-containing HIV mRNA from the nucleus to the cytoplasm. We have previously shown that splice site recognition plays an important role in Rev regulation of HIV env expression. Here we have further analyzed the effects of splice sites on HIV env expression and Rev regulation, using a simian virus 40 late replacement vector system. env expression from the vector became completely Rev-independent when an excisable intron was positioned upstream of the env region, provided that env was not recognized as an intron. Complete Rev regulation was restored either by the insertion of a 5' splice site between the intron and the env open reading frame or by deletion of the 3' splice site of the upstream intron. These results show that 5' splice sites can function as cis-acting repressor sequence (CRS) elements to retain RNA in the nucleus in the absence of Rev. They also indicate that Rev regula...

Research paper thumbnail of Regulation of human immunodeficiency virus env expression by the rev gene product

Journal of Virology, 1989

A single simian virus 40 late replacement vector which expresses both the rev and envelope (env) ... more A single simian virus 40 late replacement vector which expresses both the rev and envelope (env) genes of human immunodeficiency virus was used to examine the mechanism underlying the dependence of env gene expression on the rev protein. When rev was deleted from the vector, no envelope protein expression could be detected in transfected cells, and the levels of cytoplasmic env mRNA were dramatically reduced. In contrast to this, the levels of env RNA in total cellular RNA preparations were similar with or without rev coexpression, and analysis of nuclear RNA showed that the levels of nuclear env RNA were increased in the absence of rev. These results suggest that rev functions to regulate nuclear export of env mRNA. It was possible to restore env expression from the vector lacking rev by supplying rev in trans, provided that a cis-acting sequence was also present. This sequence was mapped to a 854-base-pair region within the env open reading frame, and it was shown that the sequenc...

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

Journal of Virology, 1999

Although most reports defining the human immunodeficiency virus type 1 (HIV-1) genomic RNA packag... more 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 a...

Research paper thumbnail of An assay to measure the activity of RNA elements and proteins that promote the expression of mRNA with retained introns

The ability to overcome cellular restrictions that exist for the export and translation of mRNAs ... more The ability to overcome cellular restrictions that exist for the export and translation of mRNAs with retained introns is a requirement for the replication of retroviruses and also for the expression of many mRNA isoforms transcribed from cellular genes. In some cases, RNA structures have been identified in the mRNA that directly interact with cellular factors to promote the export and expression of isoforms with retained introns. In other cases, a viral protein is also required to act as an adapter. In this report we describe a novel vector system that allows measurement of the ability ofcis-andtrans-acting factors to promote the export and translation of mRNA with retained introns.One reporter vector used in this system is derived from an HIV proviral clone engineered to express two different fluorescent proteins from spliced and unspliced transcripts. The ratio of fluorescent signals is a measurement of the efficiency of export and translation. A second vector utilizes a third fl...

Research paper thumbnail of Evolution of the HIV-1 RRE during natural infection reveals nucleotide changes that correlate with altered structure and increased activity over time

ABSTRACTThe HIV-1 Rev Response Element (RRE) is acis-acting RNA element characterized by multiple... more ABSTRACTThe HIV-1 Rev Response Element (RRE) is acis-acting RNA element characterized by multiple stem-loops. Binding and multimerization of the HIV Rev protein on the RRE promotes nucleocytoplasmic export of incompletely spliced mRNAs, an essential step in HIV replication. Most of our understanding of the Rev-RRE regulatory axis comes from studies of lab-adapted HIV clones. However, in human infection, HIV evolves rapidly and mechanistic studies of naturally occurring Rev and RRE sequences are essential to understanding this system. We previously described the functional activity of two RREs found in circulating viruses in a patient followed during the course of HIV infection. The “early” RRE was less functionally active than the “late” RRE despite differing in sequence by only four nucleotides. In this study, we describe the sequence, function, and structural evolution of circulating RREs in this patient using plasma samples collected over six years of untreated infection. RRE seq...

Research paper thumbnail of SR proteins: To shuttle or not to shuttle, that is the question

The Journal of cell biology, Jan 3, 2017

Serine- and arginine-rich proteins play important roles in splicing, nuclear export, and translat... more Serine- and arginine-rich proteins play important roles in splicing, nuclear export, and translation. In this issue, Botti et al. (2017. J. Cell Biol https://doi.org/10.1083/jcb.201610051) show that SRSF2 and SRSF5, previously thought to be nuclear, shuttle with messenger RNA to the cytoplasm in pluripotent P19 cells, but not in differentiated cells.

Research paper thumbnail of NXT1 (p15) Is a Crucial Cellular Cofactor in TAP-Dependent Export of Intron-Containing RNA in Mammalian Cells

Molecular and Cellular Biology, 2001

TAP, the human homologue of the yeast protein Mex67p, has been proposed to serve a role in mRNA e... more TAP, the human homologue of the yeast protein Mex67p, has been proposed to serve a role in mRNA export in mammalian cells. We have examined the ability of TAP to mediate export of Rev response element (RRE)-containing human immunodeficiency virus (HIV) RNA, a well-characterized export substrate in mammalian cells. To do this, the TAP gene was fused in frame to either RevM10 or RevΔ78–79. These proteins are nonfunctional Rev mutant proteins that can bind to HIV RNA containing the RRE in vivo but are unable to mediate the export of this RNA to the cytoplasm. However, the fusion of TAP to either of these mutant proteins gave rise to chimeric proteins that were able to complement Rev function. Significantly, cotransfection with a vector expressing NXT1 (p15), an NTF2-related cellular factor that binds to TAP, led to dramatic enhancement of the ability of the chimeric proteins to mediate RNA export. Mutant-protein analysis demonstrated that the domain necessary for nuclear export mapped ...

Research paper thumbnail of A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA

Molecular and Cellular Biology, 1997

A common feature of gene expression in all retroviruses is that unspliced, intron-containing RNA ... more A common feature of gene expression in all retroviruses is that unspliced, intron-containing RNA is exported to the cytoplasm despite the fact that cellular RNAs which contain introns are usually restricted to the nucleus. In complex retroviruses, the export of intron-containing RNA is mediated by specific viral regulatory proteins (e.g., human immunodeficiency virus type 1 [HIV-1] Rev) that bind to elements in the viral RNA. However, simpler retroviruses do not encode such regulatory proteins. Here we show that the genome of the simpler retrovirus Mason-Pfizer monkey virus (MPMV) contains an element that serves as an autonomous nuclear export signal for intron-containing RNA. This element is essential for MPMV replication; however, its function can be complemented by HIV-1 Rev and the Rev-responsive element. The element can also facilitate the export of cellular intron-containing RNA. These results suggest that the MPMV element mimics cellular RNA transport signals and mediates RNA...

[Research paper thumbnail of Inhibition of the Cellular Rev Response and HIV-1 Replication by 8-Alkyl-2-(4-pyridyl)pyrido[2,3-d pyrimidin-5(8H)-Ones](https://attachments.academia-assets.com/87790359/thumbnails/1.jpg)

Antiviral Chemistry and Chemotherapy, 1994

A high-capacity, 96-well plate assay in COS-1 cells was developed to screen for inhibitors of the... more A high-capacity, 96-well plate assay in COS-1 cells was developed to screen for inhibitors of the essential HIV-1 Rev response. The assay used Rev-induced expression and cell excretion of the p24 protein from the HIV-1 gagpol gene as a readout. Co-expression of β-galactosidase was used as a specificity control. Using this assay as a drug discovery screen, the authors discovered a series of 8-alkyl-2-(4-pyridyl)pyrido[2,3- d]pyrimidin-5(8 H)-ones that inhibited the primary Rev response in COS-1 cells with IC50s in the range 2-20 μm. These compounds also inhibited HIV-1 strain IIIB replication in human H9 cells (T-cell lymphoma) with IC50s in the same concentration range. Limited structural information suggests that alkyl substituent on N(8) influences potency of this series. These compounds might be the first reported small-molecule inhibitors of HIV-1 replication which act by inhibiting the essential Rev response; further studies in T-cells are in progress to confirm this hypothesis.

Research paper thumbnail of The HIV-1 Rev response element (RRE) adopts alternative conformations that promote different rates of virus replication

Nucleic acids research, Jan 8, 2015

The HIV Rev protein forms a complex with a 351 nucleotide sequence present in unspliced and incom... more The HIV Rev protein forms a complex with a 351 nucleotide sequence present in unspliced and incompletely spliced human immunodeficiency virus (HIV) mRNAs, the Rev response element (RRE), to recruit the cellular nuclear export receptor Crm1 and Ran-GTP. This complex facilitates nucleo-cytoplasmic export of these mRNAs. The precise secondary structure of the HIV-1 RRE has been controversial, since studies have reported alternative structures comprising either four or five stem-loops. The published structures differ only in regions that lie outside of the primary Rev binding site. Using in-gel SHAPE, we have now determined that the wt NL4-3 RRE exists as a mixture of both structures. To assess functional differences between these RRE 'conformers', we created conformationally locked mutants by site-directed mutagenesis. Using subgenomic reporters, as well as HIV replication assays, we demonstrate that the five stem-loop form of the RRE promotes greater functional Rev/RRE activit...

Research paper thumbnail of Evolutionary conservation of a molecular machinery for export and expression of mRNAs with retained introns

RNA (New York, N.Y.), 2015

Intron retention is one of the least studied forms of alternative splicing. Through the use of re... more Intron retention is one of the least studied forms of alternative splicing. Through the use of retrovirus and other model systems, it was established many years ago that mRNAs with retained introns are subject to restriction both at the level of nucleocytoplasmic export and cytoplasmic expression. It was also demonstrated that specific cis-acting elements in the mRNA could serve to bypass these restrictions. Here we show that one of these elements, the constitutive transport element (CTE), first identified in the retrovirus MPMV and subsequently in the human NXF1 gene, is a highly conserved element. Using GERP analysis, CTEs with strong primary sequence homology, predicted to display identical secondary structure, were identified in NXF genes from >30 mammalian species. CTEs were also identified in the predicted NXF1 genes of zebrafish and coelacanths. The CTE from the zebrafish NXF1 was shown to function efficiently to achieve expression of mRNA with a retained intron in human c...

Research paper thumbnail of A Long-Awaited Structure Is Rev-ealed

Viruses, 2011

It has been known for some time that the HIV Rev protein binds and oligomerizes on a well-defined... more It has been known for some time that the HIV Rev protein binds and oligomerizes on a well-defined multiple stem-loop RNA structure, named the Rev Response Element (RRE), which is present in a subset of HIV mRNAs. This binding is the first step in a pathway that overcomes a host restriction, which would otherwise prevent the export of these RNAs to the cytoplasm. Four recent publications now provide new insight into the structure of Rev and the multimeric RNA-protein complex that forms on the RRE [1-4]. Two unexpected and remarkable findings revealed in these studies are the flexibility of RNA binding that is demonstrated by the Rev arginine-rich RNA binding motif, and the way that both Rev protein and RRE contribute to the formation of the complex in a highly cooperative fashion. These studies also define the Rev dimerization and oligomerization interfaces to a resolution of 2.5Å, providing a framework necessary for further structural and functional studies. Additionally, and perhaps most importantly, they also pave the way for rational drug design, which may ultimately lead to new therapies to inhibit this essential HIV function.

Research paper thumbnail of A small element from the Mason-Pfizer monkey virusgenome makes human immunodeficiency virus type 1 expression and replicationRev-independent

Proceedings of the National Academy of Sciences, 1994

Replication of human immunodeficiency virus type 1 (HIV-1) is dependent on the viral Rev protein.... more Replication of human immunodeficiency virus type 1 (HIV-1) is dependent on the viral Rev protein. This protein acts in concert with the cis-acting rev-responsive element present in intron-containing RNAs to facilitate nuclear export of these RNAs. Here we show that a cis-acting 219-nucleotide sequence from an unrelated "simple" retrovirus, Mason-Pfizer monkey virus (MPMV), enables Rev-independent HIV-1 replication. This sequence is present in an untranslated region near the 3' end of the MPMV genome. The MPMV element is also able to efficiently substitute for Rev in expression of Gag/Pol and Env proteins from subgenomic constructs. We hypothesize that the MPMV element functions by interacting with a cellular factor that plays a role in mRNA transport analogous to that of the Rev protein. It might be possible to exploit this element in the development of an HIV vaccine.

Research paper thumbnail of U1 small nuclear RNA plays a direct role in the formation of a rev-regulated human immunodeficiency virus env mRNA that remains unspliced

Proceedings of the National Academy of Sciences, 1990

rev-regulated expression of HIV-1 envelope proteins from a simian virus 40 late replacement vecto... more rev-regulated expression of HIV-1 envelope proteins from a simian virus 40 late replacement vector was found to be dependent on the presence of a 5' splice site in the env mRNA in spite of the fact that this mRNA remains unspliced. When the 5' splice site upstream of the env open reading frame was deleted or mutated, expression of envelope protein was lost. RNA analysis of cells transfected with 5' splice-site mutants showed a dramatic reduction in the steady-state levels of env mRNA whether or not rev was present. Envelope expression could be restored in one of the 5' splice-site mutants by cotransfection with a plasmid expressing a suppressor U1 small nuclear RNA containing a compensatory mutation. These experiments show that U1 small nuclear RNA plays a direct and essential role in the formation of an unspliced RNA that is subject to regulation by rev.

Research paper thumbnail of Probing the HIV-1 Genomic RNA Trafficking Pathway and Dimerization by Genetic Recombination and Single Virion Analyses

Research paper thumbnail of Mutations in Tap Uncouple RNA Export Activity from Translocation through the Nuclear Pore Complex

Molecular Biology of the Cell, 2005

Interactions between transport receptors and phenylalanine-glycine (FG) repeats on nucleoporins d... more Interactions between transport receptors and phenylalanine-glycine (FG) repeats on nucleoporins drive the translocation of receptor-cargo complexes through nuclear pores. Tap, a transport receptor that mediates nuclear export of cellular mRNAs, contains a UBA-like and NTF2-like folds that can associate directly with FG repeats. In addition, two nuclear export sequences (NESs) within the NTF2-like region can also interact with nucleoporins. The Tap-RNA complex was shown to bind to three nucleoporins, Nup98, p62, and RanBP2, and these interactions were enhanced by Nxt1. Mutations in the Tap-UBA region abolished interactions with all three nucleoporins, whereas the effect of point mutations within the NTF2-like domain of Tap known to disrupt Nxt1 binding or nucleoporin binding were nucleoporin dependent. A mutation in any of these Tap domains was sufficient to reduce RNA export but was not sufficient to disrupt Tap interaction with the NPC in vivo or its nucleocytoplasmic shuttling. Ho...

Research paper thumbnail of Identification of a cis-acting element in human immunodeficiency virus type 2 (HIV-2) that is responsive to the HIV-1 rev and human T-cell leukemia virus types I and II rex proteins

Journal of Virology, 1990

A simian virus 40 late replacement vector encoding human immunodeficiency virus type 1 (HIV-1) gp... more A simian virus 40 late replacement vector encoding human immunodeficiency virus type 1 (HIV-1) gp120 (pGP120) was used to define a region within the HIV-2 genome that could work as a rev-responsive element (RRE). Our previous work showed that gp120 expression in this system required a functional RRE in cis and required the rev protein in trans (M.-L. Hammarskjöld, J. Heimer, B. Hammarskjöld, I. Sangwan, L. Albert, and D. Rekosh, J. Virol. 63:1959-1966, 1989). Using pGP120, we first mapped an RRE to a 1,042-base-pair (bp) Sau3a fragment in the env region of HIV-2. Both HIV-1 rev (rev1) and HIV-2 rev (rev2) could work in conjunction with this fragment. Further mapping showed that a 272-bp subfragment within the 1,042-bp region was sufficient as an RRE. Surprisingly, the smaller fragment worked only with the rev1 protein and not with its homologous rev2 protein. In addition, the rev2 protein failed to function together with the RRE from HIV-1. We also utilized this system to examine th...

Research paper thumbnail of RNA and lessons from viruses

RNA (New York, N.Y.), 2015

Research paper thumbnail of The effect of viral regulatory protein expression on gene delivery by human immunodeficiency virus type 1 vectors produced in stable packaging cell lines

Journal of virology, 1997

We describe the generation of stable human immunodeficiency virus type 1 (HIV-1)-packaging lines ... more We describe the generation of stable human immunodeficiency virus type 1 (HIV-1)-packaging lines that constitutively express high levels of HIV-1 structural proteins in either a Rev-dependent or a Rev-independent fashion. These cell lines were used to assess gene transfer by using an HIV-1 vector expressing the hygromycin B resistance gene and to study the effects of Rev, Tat, and Nef on the vector titer. The Rev-independent cell lines were created by using gag-pol and env expression vectors that contain the Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE). Vector titers approaching 10(4) CFU/ml were routinely obtained with these cell lines, as well as with the Rev-dependent cell lines, with HeLa-CD4 cells as targets. The presence of Nef and Tat in the producer cell each increased the vector titer 5- to 10-fold. Rev, on the other hand, was absolutely essential for gene transfer, unless the MPMV CTE was present in the vector. In that case, by using the Rev-indepe...

Research paper thumbnail of Human immunodeficiency virus type 1 Pr55gag and Pr160gag-pol expressed from a simian virus 40 late replacement vector are efficiently processed and assembled into viruslike particles

Journal of Virology, 1990

Human immunodeficiency virus type 1 (HIV-1) gag and pol genes were expressed by using fragments o... more Human immunodeficiency virus type 1 (HIV-1) gag and pol genes were expressed by using fragments of the BH10 clone of HIV inserted into a simian virus 40 late replacement vector. An initial construct containing the entire coding regions of gag, pol, and vif produced only minute amounts of the gag precursor, Pr55gag. However, high-level expression was obtained when an additional sequence from the env gene (the rev-responsive element) was inserted 3' of vif in the correct orientation, and rev was provided in trans from a second vector. Western immunoblot analysis of transfected cells showed the presence of large amounts of both Pr55gag and Pr160gag-pol as well as all of the expected cleavage products. Electron microscopy of thin sections of transfected cells showed a multitude of viruslike particles. Both immature particles in the process of budding and particles containing the condensed core characteristic of HIV were observed. Analysis of the released viruslike particles showed t...

Research paper thumbnail of Human immunodeficiency virus env expression becomes Rev-independent if the env region is not defined as an intron

Journal of Virology, 1994

The human immunodeficiency virus (HIV) Rev protein functions to facilitate export of intron-conta... more The human immunodeficiency virus (HIV) Rev protein functions to facilitate export of intron-containing HIV mRNA from the nucleus to the cytoplasm. We have previously shown that splice site recognition plays an important role in Rev regulation of HIV env expression. Here we have further analyzed the effects of splice sites on HIV env expression and Rev regulation, using a simian virus 40 late replacement vector system. env expression from the vector became completely Rev-independent when an excisable intron was positioned upstream of the env region, provided that env was not recognized as an intron. Complete Rev regulation was restored either by the insertion of a 5' splice site between the intron and the env open reading frame or by deletion of the 3' splice site of the upstream intron. These results show that 5' splice sites can function as cis-acting repressor sequence (CRS) elements to retain RNA in the nucleus in the absence of Rev. They also indicate that Rev regula...

Research paper thumbnail of Regulation of human immunodeficiency virus env expression by the rev gene product

Journal of Virology, 1989

A single simian virus 40 late replacement vector which expresses both the rev and envelope (env) ... more A single simian virus 40 late replacement vector which expresses both the rev and envelope (env) genes of human immunodeficiency virus was used to examine the mechanism underlying the dependence of env gene expression on the rev protein. When rev was deleted from the vector, no envelope protein expression could be detected in transfected cells, and the levels of cytoplasmic env mRNA were dramatically reduced. In contrast to this, the levels of env RNA in total cellular RNA preparations were similar with or without rev coexpression, and analysis of nuclear RNA showed that the levels of nuclear env RNA were increased in the absence of rev. These results suggest that rev functions to regulate nuclear export of env mRNA. It was possible to restore env expression from the vector lacking rev by supplying rev in trans, provided that a cis-acting sequence was also present. This sequence was mapped to a 854-base-pair region within the env open reading frame, and it was shown that the sequenc...

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

Journal of Virology, 1999

Although most reports defining the human immunodeficiency virus type 1 (HIV-1) genomic RNA packag... more 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 a...

Research paper thumbnail of An assay to measure the activity of RNA elements and proteins that promote the expression of mRNA with retained introns

The ability to overcome cellular restrictions that exist for the export and translation of mRNAs ... more The ability to overcome cellular restrictions that exist for the export and translation of mRNAs with retained introns is a requirement for the replication of retroviruses and also for the expression of many mRNA isoforms transcribed from cellular genes. In some cases, RNA structures have been identified in the mRNA that directly interact with cellular factors to promote the export and expression of isoforms with retained introns. In other cases, a viral protein is also required to act as an adapter. In this report we describe a novel vector system that allows measurement of the ability ofcis-andtrans-acting factors to promote the export and translation of mRNA with retained introns.One reporter vector used in this system is derived from an HIV proviral clone engineered to express two different fluorescent proteins from spliced and unspliced transcripts. The ratio of fluorescent signals is a measurement of the efficiency of export and translation. A second vector utilizes a third fl...

Research paper thumbnail of Evolution of the HIV-1 RRE during natural infection reveals nucleotide changes that correlate with altered structure and increased activity over time

ABSTRACTThe HIV-1 Rev Response Element (RRE) is acis-acting RNA element characterized by multiple... more ABSTRACTThe HIV-1 Rev Response Element (RRE) is acis-acting RNA element characterized by multiple stem-loops. Binding and multimerization of the HIV Rev protein on the RRE promotes nucleocytoplasmic export of incompletely spliced mRNAs, an essential step in HIV replication. Most of our understanding of the Rev-RRE regulatory axis comes from studies of lab-adapted HIV clones. However, in human infection, HIV evolves rapidly and mechanistic studies of naturally occurring Rev and RRE sequences are essential to understanding this system. We previously described the functional activity of two RREs found in circulating viruses in a patient followed during the course of HIV infection. The “early” RRE was less functionally active than the “late” RRE despite differing in sequence by only four nucleotides. In this study, we describe the sequence, function, and structural evolution of circulating RREs in this patient using plasma samples collected over six years of untreated infection. RRE seq...

Research paper thumbnail of SR proteins: To shuttle or not to shuttle, that is the question

The Journal of cell biology, Jan 3, 2017

Serine- and arginine-rich proteins play important roles in splicing, nuclear export, and translat... more Serine- and arginine-rich proteins play important roles in splicing, nuclear export, and translation. In this issue, Botti et al. (2017. J. Cell Biol https://doi.org/10.1083/jcb.201610051) show that SRSF2 and SRSF5, previously thought to be nuclear, shuttle with messenger RNA to the cytoplasm in pluripotent P19 cells, but not in differentiated cells.

Research paper thumbnail of NXT1 (p15) Is a Crucial Cellular Cofactor in TAP-Dependent Export of Intron-Containing RNA in Mammalian Cells

Molecular and Cellular Biology, 2001

TAP, the human homologue of the yeast protein Mex67p, has been proposed to serve a role in mRNA e... more TAP, the human homologue of the yeast protein Mex67p, has been proposed to serve a role in mRNA export in mammalian cells. We have examined the ability of TAP to mediate export of Rev response element (RRE)-containing human immunodeficiency virus (HIV) RNA, a well-characterized export substrate in mammalian cells. To do this, the TAP gene was fused in frame to either RevM10 or RevΔ78–79. These proteins are nonfunctional Rev mutant proteins that can bind to HIV RNA containing the RRE in vivo but are unable to mediate the export of this RNA to the cytoplasm. However, the fusion of TAP to either of these mutant proteins gave rise to chimeric proteins that were able to complement Rev function. Significantly, cotransfection with a vector expressing NXT1 (p15), an NTF2-related cellular factor that binds to TAP, led to dramatic enhancement of the ability of the chimeric proteins to mediate RNA export. Mutant-protein analysis demonstrated that the domain necessary for nuclear export mapped ...

Research paper thumbnail of A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA

Molecular and Cellular Biology, 1997

A common feature of gene expression in all retroviruses is that unspliced, intron-containing RNA ... more A common feature of gene expression in all retroviruses is that unspliced, intron-containing RNA is exported to the cytoplasm despite the fact that cellular RNAs which contain introns are usually restricted to the nucleus. In complex retroviruses, the export of intron-containing RNA is mediated by specific viral regulatory proteins (e.g., human immunodeficiency virus type 1 [HIV-1] Rev) that bind to elements in the viral RNA. However, simpler retroviruses do not encode such regulatory proteins. Here we show that the genome of the simpler retrovirus Mason-Pfizer monkey virus (MPMV) contains an element that serves as an autonomous nuclear export signal for intron-containing RNA. This element is essential for MPMV replication; however, its function can be complemented by HIV-1 Rev and the Rev-responsive element. The element can also facilitate the export of cellular intron-containing RNA. These results suggest that the MPMV element mimics cellular RNA transport signals and mediates RNA...

[Research paper thumbnail of Inhibition of the Cellular Rev Response and HIV-1 Replication by 8-Alkyl-2-(4-pyridyl)pyrido[2,3-d pyrimidin-5(8H)-Ones](https://attachments.academia-assets.com/87790359/thumbnails/1.jpg)

Antiviral Chemistry and Chemotherapy, 1994

A high-capacity, 96-well plate assay in COS-1 cells was developed to screen for inhibitors of the... more A high-capacity, 96-well plate assay in COS-1 cells was developed to screen for inhibitors of the essential HIV-1 Rev response. The assay used Rev-induced expression and cell excretion of the p24 protein from the HIV-1 gagpol gene as a readout. Co-expression of β-galactosidase was used as a specificity control. Using this assay as a drug discovery screen, the authors discovered a series of 8-alkyl-2-(4-pyridyl)pyrido[2,3- d]pyrimidin-5(8 H)-ones that inhibited the primary Rev response in COS-1 cells with IC50s in the range 2-20 μm. These compounds also inhibited HIV-1 strain IIIB replication in human H9 cells (T-cell lymphoma) with IC50s in the same concentration range. Limited structural information suggests that alkyl substituent on N(8) influences potency of this series. These compounds might be the first reported small-molecule inhibitors of HIV-1 replication which act by inhibiting the essential Rev response; further studies in T-cells are in progress to confirm this hypothesis.

Research paper thumbnail of The HIV-1 Rev response element (RRE) adopts alternative conformations that promote different rates of virus replication

Nucleic acids research, Jan 8, 2015

The HIV Rev protein forms a complex with a 351 nucleotide sequence present in unspliced and incom... more The HIV Rev protein forms a complex with a 351 nucleotide sequence present in unspliced and incompletely spliced human immunodeficiency virus (HIV) mRNAs, the Rev response element (RRE), to recruit the cellular nuclear export receptor Crm1 and Ran-GTP. This complex facilitates nucleo-cytoplasmic export of these mRNAs. The precise secondary structure of the HIV-1 RRE has been controversial, since studies have reported alternative structures comprising either four or five stem-loops. The published structures differ only in regions that lie outside of the primary Rev binding site. Using in-gel SHAPE, we have now determined that the wt NL4-3 RRE exists as a mixture of both structures. To assess functional differences between these RRE 'conformers', we created conformationally locked mutants by site-directed mutagenesis. Using subgenomic reporters, as well as HIV replication assays, we demonstrate that the five stem-loop form of the RRE promotes greater functional Rev/RRE activit...

Research paper thumbnail of Evolutionary conservation of a molecular machinery for export and expression of mRNAs with retained introns

RNA (New York, N.Y.), 2015

Intron retention is one of the least studied forms of alternative splicing. Through the use of re... more Intron retention is one of the least studied forms of alternative splicing. Through the use of retrovirus and other model systems, it was established many years ago that mRNAs with retained introns are subject to restriction both at the level of nucleocytoplasmic export and cytoplasmic expression. It was also demonstrated that specific cis-acting elements in the mRNA could serve to bypass these restrictions. Here we show that one of these elements, the constitutive transport element (CTE), first identified in the retrovirus MPMV and subsequently in the human NXF1 gene, is a highly conserved element. Using GERP analysis, CTEs with strong primary sequence homology, predicted to display identical secondary structure, were identified in NXF genes from >30 mammalian species. CTEs were also identified in the predicted NXF1 genes of zebrafish and coelacanths. The CTE from the zebrafish NXF1 was shown to function efficiently to achieve expression of mRNA with a retained intron in human c...

Research paper thumbnail of A Long-Awaited Structure Is Rev-ealed

Viruses, 2011

It has been known for some time that the HIV Rev protein binds and oligomerizes on a well-defined... more It has been known for some time that the HIV Rev protein binds and oligomerizes on a well-defined multiple stem-loop RNA structure, named the Rev Response Element (RRE), which is present in a subset of HIV mRNAs. This binding is the first step in a pathway that overcomes a host restriction, which would otherwise prevent the export of these RNAs to the cytoplasm. Four recent publications now provide new insight into the structure of Rev and the multimeric RNA-protein complex that forms on the RRE [1-4]. Two unexpected and remarkable findings revealed in these studies are the flexibility of RNA binding that is demonstrated by the Rev arginine-rich RNA binding motif, and the way that both Rev protein and RRE contribute to the formation of the complex in a highly cooperative fashion. These studies also define the Rev dimerization and oligomerization interfaces to a resolution of 2.5Å, providing a framework necessary for further structural and functional studies. Additionally, and perhaps most importantly, they also pave the way for rational drug design, which may ultimately lead to new therapies to inhibit this essential HIV function.

Research paper thumbnail of A small element from the Mason-Pfizer monkey virusgenome makes human immunodeficiency virus type 1 expression and replicationRev-independent

Proceedings of the National Academy of Sciences, 1994

Replication of human immunodeficiency virus type 1 (HIV-1) is dependent on the viral Rev protein.... more Replication of human immunodeficiency virus type 1 (HIV-1) is dependent on the viral Rev protein. This protein acts in concert with the cis-acting rev-responsive element present in intron-containing RNAs to facilitate nuclear export of these RNAs. Here we show that a cis-acting 219-nucleotide sequence from an unrelated "simple" retrovirus, Mason-Pfizer monkey virus (MPMV), enables Rev-independent HIV-1 replication. This sequence is present in an untranslated region near the 3' end of the MPMV genome. The MPMV element is also able to efficiently substitute for Rev in expression of Gag/Pol and Env proteins from subgenomic constructs. We hypothesize that the MPMV element functions by interacting with a cellular factor that plays a role in mRNA transport analogous to that of the Rev protein. It might be possible to exploit this element in the development of an HIV vaccine.

Research paper thumbnail of U1 small nuclear RNA plays a direct role in the formation of a rev-regulated human immunodeficiency virus env mRNA that remains unspliced

Proceedings of the National Academy of Sciences, 1990

rev-regulated expression of HIV-1 envelope proteins from a simian virus 40 late replacement vecto... more rev-regulated expression of HIV-1 envelope proteins from a simian virus 40 late replacement vector was found to be dependent on the presence of a 5' splice site in the env mRNA in spite of the fact that this mRNA remains unspliced. When the 5' splice site upstream of the env open reading frame was deleted or mutated, expression of envelope protein was lost. RNA analysis of cells transfected with 5' splice-site mutants showed a dramatic reduction in the steady-state levels of env mRNA whether or not rev was present. Envelope expression could be restored in one of the 5' splice-site mutants by cotransfection with a plasmid expressing a suppressor U1 small nuclear RNA containing a compensatory mutation. These experiments show that U1 small nuclear RNA plays a direct and essential role in the formation of an unspliced RNA that is subject to regulation by rev.

Research paper thumbnail of Probing the HIV-1 Genomic RNA Trafficking Pathway and Dimerization by Genetic Recombination and Single Virion Analyses

Research paper thumbnail of Mutations in Tap Uncouple RNA Export Activity from Translocation through the Nuclear Pore Complex

Molecular Biology of the Cell, 2005

Interactions between transport receptors and phenylalanine-glycine (FG) repeats on nucleoporins d... more Interactions between transport receptors and phenylalanine-glycine (FG) repeats on nucleoporins drive the translocation of receptor-cargo complexes through nuclear pores. Tap, a transport receptor that mediates nuclear export of cellular mRNAs, contains a UBA-like and NTF2-like folds that can associate directly with FG repeats. In addition, two nuclear export sequences (NESs) within the NTF2-like region can also interact with nucleoporins. The Tap-RNA complex was shown to bind to three nucleoporins, Nup98, p62, and RanBP2, and these interactions were enhanced by Nxt1. Mutations in the Tap-UBA region abolished interactions with all three nucleoporins, whereas the effect of point mutations within the NTF2-like domain of Tap known to disrupt Nxt1 binding or nucleoporin binding were nucleoporin dependent. A mutation in any of these Tap domains was sufficient to reduce RNA export but was not sufficient to disrupt Tap interaction with the NPC in vivo or its nucleocytoplasmic shuttling. Ho...