Richard Fridell - Academia.edu (original) (raw)

Papers by Richard Fridell

Research paper thumbnail of Historic distribution, current status and range extension of Bufo boreas in Utah

Herpetological review, 1995

Research paper thumbnail of Nuclear import of hnRNP A1 is mediated by a novel cellular cofactor related to karyopherin-beta

Journal of Cell Science, 1997

Heterogeneous nuclear ribonucleoprotein A1 contains a sequence, termed M9, that functions as a po... more Heterogeneous nuclear ribonucleoprotein A1 contains a sequence, termed M9, that functions as a potent nuclear localization signal (NLS) yet bears no similarity to the well-defined basic class of NLSs. Here, we report the identification of a novel human protein, termed MIP, that binds M9 specifically both in vivo and in vitro yet fails to interact with non-functional M9 point mutants. Of note, the 101 kDa MIP protein bears significant homology to human karyopherin/importin-beta, a protein known to mediate the function of basic NLSs. The in vitro nuclear import of a protein substrate containing the M9 NLS was found to be dependent on provision of the MIP protein in trans. Cytoplasmic microinjection of a truncated form of MIP that retains the M9 binding site blocked the in vivo nuclear import of a substrate containing the M9 NLS yet failed to affect the import of a similar substrate bearing a basic NLS. These data indicate that nuclear import of hnRNP A1 is mediated by a novel cellular...

Research paper thumbnail of Murine CXCR-4 is a functional coreceptor for T-cell-tropic and dual-tropic strains of human immunodeficiency virus type 1

Journal of Virology, 1997

The human chemokine receptor hCXCR-4 serves as a coreceptor for T-cell-tropic (T-tropic) and dual... more The human chemokine receptor hCXCR-4 serves as a coreceptor for T-cell-tropic (T-tropic) and dual-tropic strains of human immunodeficiency virus type 1 (HIV-1). We have isolated a homolog of hCXCR-4 from a murine T-cell cDNA library and have examined its ability to function as an HIV-1 coreceptor. mCXCR-4 was found to be 91% identical to the human receptor at the amino acid level, with sequence differences concentrated in extracellular domains. Surprisingly, coexpression of both hCD4 and mCXCR-4 on either simian or murine cell lines rendered them permissive for HIV-1-induced cell fusion, indicating that mCXCR-4 is a functional HIV-1 coreceptor. As with hCXCR-4, coreceptor function was restricted to T-tropic and dual-tropic HIV-1 strains. Ribonuclease protection analysis indicated that mCXCR-4 mRNA was expressed in only two of six murine cell lines tested. In contrast, Northern blot analysis of human and mouse tissues revealed that CXCR-4 is widely expressed in both species in vivo. ...

Research paper thumbnail of Identification of the activation domain of equine infectious anemia virus rev

Journal of Virology, 1993

Several members of the lentivirus family of complex retroviruses have been shown to encode protei... more Several members of the lentivirus family of complex retroviruses have been shown to encode proteins that are functionally equivalent to the Rev posttranscriptional regulatory protein of human immunodeficiency virus type 1 (HIV-1). Furthermore, the domain organization of HIV-1 Rev, featuring a highly basic N-terminal RNA binding domain and a leucin-rich C-terminal effector domain, has also been shown to be highly conserved among Rev proteins derived from not only the primate but also the ovine and caprine lentiviruses. Although it has therefore appeared highly probable that the lentivirus equine infectious anemia virus (EIAV) also encodes a Rev, the predicted amino acid sequence of this putative EIAV regulatory protein does not display any evident homology to the basic and leucine-rich motifs characteristic of other known Rev proteins. By fusion of different segments of the proposed EIAV Rev protein to the well-defined RNA binding domain of either HIV-1 or visna virus Rev, we have id...

Research paper thumbnail of Genetic evidence that the Tat proteins of human immunodeficiency virus types 1 and 2 can multimerize in the eukaryotic cell nucleus

Journal of Virology, 1993

The formation of dimers or higher-order multimers is critical to the biological activity of many ... more The formation of dimers or higher-order multimers is critical to the biological activity of many eukaryotic regulatory proteins. However, biochemical analyses of the multimerization capacity of the Tat trans activator of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) have yielded contradictory results. We used the two-hybrid genetic assay for protein-protein interactions in the eukaryote Saccharomyces cerevisiae (S. Fields and O.-K. Song, Nature [London] 340:245-246, 1989) to examine the multimerization of Tat in vivo. Both HIV-1 and HIV-2 Tat are shown to form specific homo- but not heteromultimers in the yeast cell nucleus. Mutational analysis indicates a critical role for the essential core motif of Tat in mediating this interaction but demonstrates that efficient Tat multimerization does not require an intact cysteine motif. These data raise the possibility that the multimerization of Tat may be important for Tat function in higher eukaryotes.

Research paper thumbnail of Aquatic survey of the lower Escalante River, Glen Canyon National Recreation Area, Utah, June 22-26, 1998

Open-File Report, 1999

This open-file report provides a discription of one field trip. It was not intended to represent ... more This open-file report provides a discription of one field trip. It was not intended to represent an extensive survey effort nor a complete analysis of field data. This report represents a repository of field data, observations and discriptions of ecological conditions in June 1998, and provides some recommendations for resource managers.

Research paper thumbnail of Synergistic enhancement of both initiation and elongation by acidic transcription activation domains

The EMBO Journal, 1996

The effects of activation domain synergy on transcription initiation and elongation have been exa... more The effects of activation domain synergy on transcription initiation and elongation have been examined utilizing a system that permits the targeting of a defined number of activation modules to promoter DNA. As predicted, incremental increases in targeted activation potential were found to result in corresponding increases in transcription initiation. Surprisingly, however, transcriptional processivity, and hence mRNA synthesis, required a threshold level of activation domain synergy that exceeded the level required for at least modest levels of transcription initiation. The degree to which transcriptional processivity was enhanced was shown to depend on the quantity of activation modules targeted to the promoter DNA, rather than the quality. While the RNA-sequence specific HIV-1 Tat trans-activator was also shown to enhance processivity in this assay system, Tat differed from DNA-sequence specific activation domains in exerting a more dramatic effect on the efficiency of transcript elongation.

Research paper thumbnail of A nuclear role for the Fragile X mental retardation protein

The EMBO Journal, 1996

Fragile X syndrome results from lack of expression of a functional form of Fragile X mental retar... more Fragile X syndrome results from lack of expression of a functional form of Fragile X mental retardation protein (FMRP), a cytoplasmic RNA-binding protein of uncertain function. Here, we report that FMRP contains a nuclear export signal (NES) that is similar to the NES recently identified in the Rev regulatory protein of human immunodeficiency virus type 1 (HIV-1). Mutation of this FMRP NES results in mislocalization of FMRP to the cell nucleus. The FMRP NES is encoded within exon 14 of the FMRJ gene, thus explaining the aberrant nuclear localization of a natural isoform of FMRP that lacks this exon. The NES of FMRP can substitute fully for the Rev NES in mediating Rev-dependent nuclear RNA export and specifically binds a nucleoporin-like cellular cofactor that has been shown to mediate Rev NES function. Together, these findings demonstrate that the normal function of FMRP involves entry into the nucleus followed by export via a pathway that is identical to the one utilized by HIV-1 Rev. In addition, these data raise the possibility that FMRP could play a role in mediating the nuclear export of its currently undefined cellular RNA target(s).

Research paper thumbnail of Structure and transcription of the Drosophila melanogaster vermilion gene and several mutant alleles

Molecular and Cellular Biology, 1990

The nucleotide sequence and intron-exon structure of the Drosophila melanogaster vermilion (v) ge... more The nucleotide sequence and intron-exon structure of the Drosophila melanogaster vermilion (v) gene have been determined. In addition, the sites of several mutations and the effects of these mutations on transcription have been examined. The major v mRNA is generated upon splicing six exons of lengths (5' to 3') 83, 161, 134, 607, 94, and 227 nucleotides (nt). A minor species of v mRNA is initiated at an upstream site and has a 5' exon of at least 152 nt which overlaps the region included in the 83-nt exon of the major v RNA. The three v mutations, v1, v2, and vk, which can be suppressed by mutations at suppressor of sable, su(s), are insertions of transposon 412 at the same position in exon 1, 36 nt downstream of the major transcription initiation site. Despite the 7.5-kilobase insertion in these v alleles, a reduced level of wild-type-sized mRNA accumulates in suppressed mutant strains. The structure and transcription of several unsuppressible v alleles have also been ...

Research paper thumbnail of Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization-selection assay

Molecular and Cellular Biology, 1996

The Rex protein of human T-cell leukemia virus type 1, like the functionally equivalent Rev prote... more The Rex protein of human T-cell leukemia virus type 1, like the functionally equivalent Rev protein of human immunodeficiency virus type 1, contains a leucine-rich activation domain that specifically interacts with the human nucleoporin-like Rab/hRIP cofactor. Here, this Rex sequence is shown to function also as a protein nuclear export signal (NES). Rex sequence libraries containing randomized forms of the activation domain/NES were screened for retention of the ability to bind Rab/hRIP by using the yeast two-hybrid assay. While the selected sequences differed widely in primary sequence, all were functional as Rex activation domains. In contrast, randomized sequences that failed to bind Rab/hRIP lacked Rex activity. The selected sequences included one with homology to the Rev activation domain/NES and a second that was similar to the NES found in the cellular protein kinase inhibitor alpha. A highly variant, yet fully active, activation domain sequence selected on the basis of Rab/...

Research paper thumbnail of Evidence for a role of the Drosophila melanogaster suppressor of sable gene in the pre-mRNA splicing pathway

Molecular and Cellular Biology, 1994

Recessive mutations of the Drosophila melanogaster suppressor of sable [su(s)] gene result in ele... more Recessive mutations of the Drosophila melanogaster suppressor of sable [su(s)] gene result in elevated accumulation of RNA from vermilion (v) mutant alleles that have an insertion of the 7.5-kb retrotransposon 412 in the first exon of the v gene. During transcription of such a v mutant gene, the 412 sequences are incorporated into the primary transcripts and are subsequently removed by splicing at cryptic sites within 412 sequences. In a su(s)+ background, the level of these unusually spliced transcripts is exceedingly low, and su(s) mutations increase their accumulation. We previously proposed that v RNA levels are elevated in su(s) mutants because of increased recognition of the cryptic splice sites, and the aim of this study was to test this hypothesis. We generated a v mutant derivative with a smaller 412 insertion, introduced alterations into the 412-associated splice sites, and examined the effect of su(s) mutations on expression of these derivatives after germ line transforma...

Research paper thumbnail of DFNB3 Families and Shaker-2 Mice: Mutations in an Unconventional Myosin, MYO 15

Advances in Oto-Rhino-Laryngology, 2000

Research paper thumbnail of Identification of a Novel Human Zinc Finger Protein That Specifically Interacts with the Activation Domain of Lentiviral Tat Proteins

Research paper thumbnail of HIV-1-induced cell fusion is mediated by multiple regions within both the viral envelope and the CCR-5 co-receptor

The EMBO Journal, 1997

Although the human hCCR-5 chemokine receptor can HIV-1 isolate able to infect both macrophages an... more Although the human hCCR-5 chemokine receptor can HIV-1 isolate able to infect both macrophages and T-cell serve as a co-receptor for both M-tropic (ADA and lines is observed, and these are referred to as dual tropic BaL) and dual-tropic (89.6) strains of human immuno-(Collman et al., 1992; Simmons et al., 1996). deficiency virus type 1 (HIV-1), the closely related Mapping of determinants of HIV-1 tropism demonmouse mCCR-5 homolog is inactive. We used chimeric strated that these were located in the viral envelope protein hCCR-5-mCCR-5 receptor molecules to examine the and were concentrated in the short V3 loop of envelope functional importance of the three extracellular (O'Brien et al., 1990; Hwang et al., 1991; Shioda et al., domains of hCCR-5 that differ in sequence from their 1991; Westervelt et al., 1991). It was therefore proposed mCCR-5 equivalents. While this analysis revealed that that infection by HIV-1 required a co-receptor, in addition all three of these extracellular domains could participto CD4, that was present on human but not on animal ate in the functional interaction with HIV-1 envelope, cells. In addition, it appeared that at least two distinct coclear differences were observed when different HIV-1 receptors must exist, one for T-tropic isolates and a strains were analyzed. Thus, while the ADA HIV-1 second specific for M-tropic isolates. Recent evidence has isolate could effectively utilize chimeric human-mouse validated this hypothesis and has identified the CXCR-4 CCR-5 chimeras containing any single human extrachemokine receptor, also termed fusin, as the predominant cellular domain, the BaL isolate required any two T-tropic HIV-1 co-receptor and the CCR-5 chemokine human extracellular sequences while the 89.6 isolate receptor as the dominant M-tropic co-receptor (Alkhatib would only interact effectively with chimeras con

Research paper thumbnail of Association of Unconventional Myosin MYO15 Mutations with Human Nonsyndromic Deafness DFNB3

Science, 1998

DFNB3 , a locus for nonsyndromic sensorineural recessive deafness, maps to a 3-centimorgan interv... more DFNB3 , a locus for nonsyndromic sensorineural recessive deafness, maps to a 3-centimorgan interval on human chromosome 17p11.2, a region that shows conserved synteny with mouse shaker-2 . A human unconventional myosin gene, MYO15 , was identified by combining functional and positional cloning approaches in searching for shaker-2 and DFNB3 . MYO15 has at least 50 exons spanning 36 kilobases. Sequence analyses of these exons in affected individuals from three unrelated DFNB3 families revealed two missense mutations and one nonsense mutation that cosegregated with congenital recessive deafness.

Research paper thumbnail of Amphibian transcription factor IIIA proteins contain a sequence element functionally equivalent to the nuclear export signal of human immunodeficiency virus type 1 Rev

Proceedings of the National Academy of Sciences, 1996

The human immunodeficiency virus type 1 (HIV-1) Rev protein is required for nuclear export of lat... more The human immunodeficiency virus type 1 (HIV-1) Rev protein is required for nuclear export of late HIV-1 mRNAs. This function is dependent on the mutationally defined Rev activation domain, which also forms a potent nuclear export signal. Transcription factor IIIA (TFIIIA) binds to 5S rRNA transcripts and this interaction has been proposed to play a role in the efficient nuclear export of 5S rRNA in amphibian oocytes. Here it is reported that amphibian TFIIIA proteins contain a sequence element with homology to the Rev activation domain that effectively substitutes for this domain in inducing the nuclear export of late HIV-1 mRNAs. It is further demonstrated that this TFIIIA sequence element functions as a protein nuclear export signal in both human cells and frog oocytes. Thus, this shared protein motif may play an analogous role in mediating the nuclear export of both late HIV-1 RNAs and 5S rRNA transcripts.

Research paper thumbnail of Nuclear export of late HIV-1 mRNAs occurs via a cellular protein export pathway

Proceedings of the National Academy of Sciences, 1996

The Rev protein of HIV-1 is essential for the nuclear export of incompletely spliced viral mRNAs.... more The Rev protein of HIV-1 is essential for the nuclear export of incompletely spliced viral mRNAs. This action depends on the mutationally defined Rev activation domain, which both binds the nucleoporin-like human cellular cofactor Rab/hRIP and also functions as a nuclear export signal. Protein kinase inhibitor alpha (PKI) also contains a potent nuclear export signal. However, PKI plays no role in nuclear RNA export and instead induces the nuclear export of a specific protein target, the catalytic subunit of cAMP-dependent protein kinase. Here, it is demonstrated that the nuclear export signal of PKI not only binds the Rab/hRIP cofactor specifically but also can effectively substitute for the Rev activation domain in mediating the nuclear export of HIV-1 mRNAs. We conclude that HIV-1 Rev and PKI act through an identical nuclear export pathway and that Rev, rather than using a dedicated RNA export pathway, is instead acting as an adaptor that allows viral mRNAs to access a cellular pr...

Research paper thumbnail of Distinct Functions of NS5A in Hepatitis C Virus RNA Replication Uncovered by Studies with the NS5A Inhibitor BMS-790052

Journal of Virology, 2011

BMS-790052, targeting nonstructural protein 5A (NS5A), is the most potent hepatitis C virus (HCV)... more BMS-790052, targeting nonstructural protein 5A (NS5A), is the most potent hepatitis C virus (HCV) inhibitor described to date. It is highly effective against genotype 1 replicons and also displays robust genotype 1 anti-HCV activity in the clinic (M. Gao et al., Nature 465:96-100, 2010). BMS-790052 inhibits genotype 2a JFH1 replicon cells and cell culture infectious virus with 50% effective concentrations (EC 50 s) of 46.8 and 16.1 pM, respectively. Resistance selection studies with the JFH1 replicon and virus systems identified drug-induced mutations within the N-terminal region of NS5A. F28S, L31M, C92R, and Y93H were the major resistance mutations identified; the impact of these mutations on inhibitor sensitivity between the replicon and virus was very similar. The C92R and Y93H mutations negatively impacted fitness of the JFH1 virus. Second-site replacements at NS5A residue 30 (K30E/Q) restored efficient replication of the C92R viral variant, thus demonstrating a genetic interac...

Research paper thumbnail of Intragenic Complementation of Hepatitis C Virus NS5A RNA Replication-Defective Alleles

Journal of Virology, 2012

Hepatitis C virus NS5A has three structural domains, is required for RNA replication and virion a... more Hepatitis C virus NS5A has three structural domains, is required for RNA replication and virion assembly, and exists in hypo- and hyperphosphorylated forms. Accumulated data suggest that phosphorylation is involved in modulating NS5A functions. We performed a mutational analysis of highly conserved serine residues in the linker region between domains I and II of genotype 2a JFH1 NS5A. As with genotype 1b Con1 NS5A, we found that specific serine residues were important for efficient hyperphosphorylation of JFH1 NS5A. However, in contrast with Con1 replicons, we observed a positive correlation between hyperphosphorylation and JFH1 replicon replication. We previously demonstrated trans -complementation of a hyperphosphorylation-deficient, replication-defective JFH1 replicon. Our results suggested that the defective NS5A encoded by this replicon, while lacking one NS5A function, was capable of performing a separate replication function. In this report, we examined an additional set of r...

Research paper thumbnail of 1424 Synergistic Interactions of HCV NS5A Replication Complex Inhibitors Sensitize Resistant Variants and Enhance the Efficacy of Daclatasvir (DCV, BMS-790052) in Vitro and in Vivo

Journal of Hepatology, 2013

LATE BREAKING ABSTRACTS 13.6±1.8 g/dL [8.8-17.5], respectively. After 12W, a complete early virol... more LATE BREAKING ABSTRACTS 13.6±1.8 g/dL [8.8-17.5], respectively. After 12W, a complete early virological response was obtained in 34 (83%) boceprevir patients and in 35 (61%) telaprevir patients (p = 0.026). Among 17 boceprevir and 16 telaprevir patients, 14 (82%) and 7 (43%) achieved an end of treatment response (EOT) with an undetetectable viral load, respectively (p = 0.032). Among 9 boceprevir and 5 telaprevir patients, 6 and 1 achieved SVR12, respectively. Among 6 patients in the boceprevir group, 3 achieved SVR24. In the telaprevir group, 29 patients discontinued therapy (serious adverse events, n = 13; virological breakthrough, n = 6; non-response, n = 9). In the boceprevir group, 14 patients discontinued therapy (serious adverse events, n = 5; virological breakthrough, n = 2; non-response, n = 4; retransplantation, n = 1). Four patients died in a context of infectious disorders: boceprevir, n = 2 (W20/W24); telaprevir, n = 2 (W2/W9). The most common side effect was anemia in 85% of patients: 95% and 96% in boceprevir and telaprevir groups received erythropoietin alone or combined with ribavirin dose reduction. Conclusion: In liver transplanted patients, EOT rate was 82% and 38% with boceprevir and telaprevir, respectively. Among the overall population, 44% of patients discontinued therapy because of treatment failure or occurrence of serious adverse events.

Research paper thumbnail of Historic distribution, current status and range extension of Bufo boreas in Utah

Herpetological review, 1995

Research paper thumbnail of Nuclear import of hnRNP A1 is mediated by a novel cellular cofactor related to karyopherin-beta

Journal of Cell Science, 1997

Heterogeneous nuclear ribonucleoprotein A1 contains a sequence, termed M9, that functions as a po... more Heterogeneous nuclear ribonucleoprotein A1 contains a sequence, termed M9, that functions as a potent nuclear localization signal (NLS) yet bears no similarity to the well-defined basic class of NLSs. Here, we report the identification of a novel human protein, termed MIP, that binds M9 specifically both in vivo and in vitro yet fails to interact with non-functional M9 point mutants. Of note, the 101 kDa MIP protein bears significant homology to human karyopherin/importin-beta, a protein known to mediate the function of basic NLSs. The in vitro nuclear import of a protein substrate containing the M9 NLS was found to be dependent on provision of the MIP protein in trans. Cytoplasmic microinjection of a truncated form of MIP that retains the M9 binding site blocked the in vivo nuclear import of a substrate containing the M9 NLS yet failed to affect the import of a similar substrate bearing a basic NLS. These data indicate that nuclear import of hnRNP A1 is mediated by a novel cellular...

Research paper thumbnail of Murine CXCR-4 is a functional coreceptor for T-cell-tropic and dual-tropic strains of human immunodeficiency virus type 1

Journal of Virology, 1997

The human chemokine receptor hCXCR-4 serves as a coreceptor for T-cell-tropic (T-tropic) and dual... more The human chemokine receptor hCXCR-4 serves as a coreceptor for T-cell-tropic (T-tropic) and dual-tropic strains of human immunodeficiency virus type 1 (HIV-1). We have isolated a homolog of hCXCR-4 from a murine T-cell cDNA library and have examined its ability to function as an HIV-1 coreceptor. mCXCR-4 was found to be 91% identical to the human receptor at the amino acid level, with sequence differences concentrated in extracellular domains. Surprisingly, coexpression of both hCD4 and mCXCR-4 on either simian or murine cell lines rendered them permissive for HIV-1-induced cell fusion, indicating that mCXCR-4 is a functional HIV-1 coreceptor. As with hCXCR-4, coreceptor function was restricted to T-tropic and dual-tropic HIV-1 strains. Ribonuclease protection analysis indicated that mCXCR-4 mRNA was expressed in only two of six murine cell lines tested. In contrast, Northern blot analysis of human and mouse tissues revealed that CXCR-4 is widely expressed in both species in vivo. ...

Research paper thumbnail of Identification of the activation domain of equine infectious anemia virus rev

Journal of Virology, 1993

Several members of the lentivirus family of complex retroviruses have been shown to encode protei... more Several members of the lentivirus family of complex retroviruses have been shown to encode proteins that are functionally equivalent to the Rev posttranscriptional regulatory protein of human immunodeficiency virus type 1 (HIV-1). Furthermore, the domain organization of HIV-1 Rev, featuring a highly basic N-terminal RNA binding domain and a leucin-rich C-terminal effector domain, has also been shown to be highly conserved among Rev proteins derived from not only the primate but also the ovine and caprine lentiviruses. Although it has therefore appeared highly probable that the lentivirus equine infectious anemia virus (EIAV) also encodes a Rev, the predicted amino acid sequence of this putative EIAV regulatory protein does not display any evident homology to the basic and leucine-rich motifs characteristic of other known Rev proteins. By fusion of different segments of the proposed EIAV Rev protein to the well-defined RNA binding domain of either HIV-1 or visna virus Rev, we have id...

Research paper thumbnail of Genetic evidence that the Tat proteins of human immunodeficiency virus types 1 and 2 can multimerize in the eukaryotic cell nucleus

Journal of Virology, 1993

The formation of dimers or higher-order multimers is critical to the biological activity of many ... more The formation of dimers or higher-order multimers is critical to the biological activity of many eukaryotic regulatory proteins. However, biochemical analyses of the multimerization capacity of the Tat trans activator of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) have yielded contradictory results. We used the two-hybrid genetic assay for protein-protein interactions in the eukaryote Saccharomyces cerevisiae (S. Fields and O.-K. Song, Nature [London] 340:245-246, 1989) to examine the multimerization of Tat in vivo. Both HIV-1 and HIV-2 Tat are shown to form specific homo- but not heteromultimers in the yeast cell nucleus. Mutational analysis indicates a critical role for the essential core motif of Tat in mediating this interaction but demonstrates that efficient Tat multimerization does not require an intact cysteine motif. These data raise the possibility that the multimerization of Tat may be important for Tat function in higher eukaryotes.

Research paper thumbnail of Aquatic survey of the lower Escalante River, Glen Canyon National Recreation Area, Utah, June 22-26, 1998

Open-File Report, 1999

This open-file report provides a discription of one field trip. It was not intended to represent ... more This open-file report provides a discription of one field trip. It was not intended to represent an extensive survey effort nor a complete analysis of field data. This report represents a repository of field data, observations and discriptions of ecological conditions in June 1998, and provides some recommendations for resource managers.

Research paper thumbnail of Synergistic enhancement of both initiation and elongation by acidic transcription activation domains

The EMBO Journal, 1996

The effects of activation domain synergy on transcription initiation and elongation have been exa... more The effects of activation domain synergy on transcription initiation and elongation have been examined utilizing a system that permits the targeting of a defined number of activation modules to promoter DNA. As predicted, incremental increases in targeted activation potential were found to result in corresponding increases in transcription initiation. Surprisingly, however, transcriptional processivity, and hence mRNA synthesis, required a threshold level of activation domain synergy that exceeded the level required for at least modest levels of transcription initiation. The degree to which transcriptional processivity was enhanced was shown to depend on the quantity of activation modules targeted to the promoter DNA, rather than the quality. While the RNA-sequence specific HIV-1 Tat trans-activator was also shown to enhance processivity in this assay system, Tat differed from DNA-sequence specific activation domains in exerting a more dramatic effect on the efficiency of transcript elongation.

Research paper thumbnail of A nuclear role for the Fragile X mental retardation protein

The EMBO Journal, 1996

Fragile X syndrome results from lack of expression of a functional form of Fragile X mental retar... more Fragile X syndrome results from lack of expression of a functional form of Fragile X mental retardation protein (FMRP), a cytoplasmic RNA-binding protein of uncertain function. Here, we report that FMRP contains a nuclear export signal (NES) that is similar to the NES recently identified in the Rev regulatory protein of human immunodeficiency virus type 1 (HIV-1). Mutation of this FMRP NES results in mislocalization of FMRP to the cell nucleus. The FMRP NES is encoded within exon 14 of the FMRJ gene, thus explaining the aberrant nuclear localization of a natural isoform of FMRP that lacks this exon. The NES of FMRP can substitute fully for the Rev NES in mediating Rev-dependent nuclear RNA export and specifically binds a nucleoporin-like cellular cofactor that has been shown to mediate Rev NES function. Together, these findings demonstrate that the normal function of FMRP involves entry into the nucleus followed by export via a pathway that is identical to the one utilized by HIV-1 Rev. In addition, these data raise the possibility that FMRP could play a role in mediating the nuclear export of its currently undefined cellular RNA target(s).

Research paper thumbnail of Structure and transcription of the Drosophila melanogaster vermilion gene and several mutant alleles

Molecular and Cellular Biology, 1990

The nucleotide sequence and intron-exon structure of the Drosophila melanogaster vermilion (v) ge... more The nucleotide sequence and intron-exon structure of the Drosophila melanogaster vermilion (v) gene have been determined. In addition, the sites of several mutations and the effects of these mutations on transcription have been examined. The major v mRNA is generated upon splicing six exons of lengths (5' to 3') 83, 161, 134, 607, 94, and 227 nucleotides (nt). A minor species of v mRNA is initiated at an upstream site and has a 5' exon of at least 152 nt which overlaps the region included in the 83-nt exon of the major v RNA. The three v mutations, v1, v2, and vk, which can be suppressed by mutations at suppressor of sable, su(s), are insertions of transposon 412 at the same position in exon 1, 36 nt downstream of the major transcription initiation site. Despite the 7.5-kilobase insertion in these v alleles, a reduced level of wild-type-sized mRNA accumulates in suppressed mutant strains. The structure and transcription of several unsuppressible v alleles have also been ...

Research paper thumbnail of Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization-selection assay

Molecular and Cellular Biology, 1996

The Rex protein of human T-cell leukemia virus type 1, like the functionally equivalent Rev prote... more The Rex protein of human T-cell leukemia virus type 1, like the functionally equivalent Rev protein of human immunodeficiency virus type 1, contains a leucine-rich activation domain that specifically interacts with the human nucleoporin-like Rab/hRIP cofactor. Here, this Rex sequence is shown to function also as a protein nuclear export signal (NES). Rex sequence libraries containing randomized forms of the activation domain/NES were screened for retention of the ability to bind Rab/hRIP by using the yeast two-hybrid assay. While the selected sequences differed widely in primary sequence, all were functional as Rex activation domains. In contrast, randomized sequences that failed to bind Rab/hRIP lacked Rex activity. The selected sequences included one with homology to the Rev activation domain/NES and a second that was similar to the NES found in the cellular protein kinase inhibitor alpha. A highly variant, yet fully active, activation domain sequence selected on the basis of Rab/...

Research paper thumbnail of Evidence for a role of the Drosophila melanogaster suppressor of sable gene in the pre-mRNA splicing pathway

Molecular and Cellular Biology, 1994

Recessive mutations of the Drosophila melanogaster suppressor of sable [su(s)] gene result in ele... more Recessive mutations of the Drosophila melanogaster suppressor of sable [su(s)] gene result in elevated accumulation of RNA from vermilion (v) mutant alleles that have an insertion of the 7.5-kb retrotransposon 412 in the first exon of the v gene. During transcription of such a v mutant gene, the 412 sequences are incorporated into the primary transcripts and are subsequently removed by splicing at cryptic sites within 412 sequences. In a su(s)+ background, the level of these unusually spliced transcripts is exceedingly low, and su(s) mutations increase their accumulation. We previously proposed that v RNA levels are elevated in su(s) mutants because of increased recognition of the cryptic splice sites, and the aim of this study was to test this hypothesis. We generated a v mutant derivative with a smaller 412 insertion, introduced alterations into the 412-associated splice sites, and examined the effect of su(s) mutations on expression of these derivatives after germ line transforma...

Research paper thumbnail of DFNB3 Families and Shaker-2 Mice: Mutations in an Unconventional Myosin, MYO 15

Advances in Oto-Rhino-Laryngology, 2000

Research paper thumbnail of Identification of a Novel Human Zinc Finger Protein That Specifically Interacts with the Activation Domain of Lentiviral Tat Proteins

Research paper thumbnail of HIV-1-induced cell fusion is mediated by multiple regions within both the viral envelope and the CCR-5 co-receptor

The EMBO Journal, 1997

Although the human hCCR-5 chemokine receptor can HIV-1 isolate able to infect both macrophages an... more Although the human hCCR-5 chemokine receptor can HIV-1 isolate able to infect both macrophages and T-cell serve as a co-receptor for both M-tropic (ADA and lines is observed, and these are referred to as dual tropic BaL) and dual-tropic (89.6) strains of human immuno-(Collman et al., 1992; Simmons et al., 1996). deficiency virus type 1 (HIV-1), the closely related Mapping of determinants of HIV-1 tropism demonmouse mCCR-5 homolog is inactive. We used chimeric strated that these were located in the viral envelope protein hCCR-5-mCCR-5 receptor molecules to examine the and were concentrated in the short V3 loop of envelope functional importance of the three extracellular (O'Brien et al., 1990; Hwang et al., 1991; Shioda et al., domains of hCCR-5 that differ in sequence from their 1991; Westervelt et al., 1991). It was therefore proposed mCCR-5 equivalents. While this analysis revealed that that infection by HIV-1 required a co-receptor, in addition all three of these extracellular domains could participto CD4, that was present on human but not on animal ate in the functional interaction with HIV-1 envelope, cells. In addition, it appeared that at least two distinct coclear differences were observed when different HIV-1 receptors must exist, one for T-tropic isolates and a strains were analyzed. Thus, while the ADA HIV-1 second specific for M-tropic isolates. Recent evidence has isolate could effectively utilize chimeric human-mouse validated this hypothesis and has identified the CXCR-4 CCR-5 chimeras containing any single human extrachemokine receptor, also termed fusin, as the predominant cellular domain, the BaL isolate required any two T-tropic HIV-1 co-receptor and the CCR-5 chemokine human extracellular sequences while the 89.6 isolate receptor as the dominant M-tropic co-receptor (Alkhatib would only interact effectively with chimeras con

Research paper thumbnail of Association of Unconventional Myosin MYO15 Mutations with Human Nonsyndromic Deafness DFNB3

Science, 1998

DFNB3 , a locus for nonsyndromic sensorineural recessive deafness, maps to a 3-centimorgan interv... more DFNB3 , a locus for nonsyndromic sensorineural recessive deafness, maps to a 3-centimorgan interval on human chromosome 17p11.2, a region that shows conserved synteny with mouse shaker-2 . A human unconventional myosin gene, MYO15 , was identified by combining functional and positional cloning approaches in searching for shaker-2 and DFNB3 . MYO15 has at least 50 exons spanning 36 kilobases. Sequence analyses of these exons in affected individuals from three unrelated DFNB3 families revealed two missense mutations and one nonsense mutation that cosegregated with congenital recessive deafness.

Research paper thumbnail of Amphibian transcription factor IIIA proteins contain a sequence element functionally equivalent to the nuclear export signal of human immunodeficiency virus type 1 Rev

Proceedings of the National Academy of Sciences, 1996

The human immunodeficiency virus type 1 (HIV-1) Rev protein is required for nuclear export of lat... more The human immunodeficiency virus type 1 (HIV-1) Rev protein is required for nuclear export of late HIV-1 mRNAs. This function is dependent on the mutationally defined Rev activation domain, which also forms a potent nuclear export signal. Transcription factor IIIA (TFIIIA) binds to 5S rRNA transcripts and this interaction has been proposed to play a role in the efficient nuclear export of 5S rRNA in amphibian oocytes. Here it is reported that amphibian TFIIIA proteins contain a sequence element with homology to the Rev activation domain that effectively substitutes for this domain in inducing the nuclear export of late HIV-1 mRNAs. It is further demonstrated that this TFIIIA sequence element functions as a protein nuclear export signal in both human cells and frog oocytes. Thus, this shared protein motif may play an analogous role in mediating the nuclear export of both late HIV-1 RNAs and 5S rRNA transcripts.

Research paper thumbnail of Nuclear export of late HIV-1 mRNAs occurs via a cellular protein export pathway

Proceedings of the National Academy of Sciences, 1996

The Rev protein of HIV-1 is essential for the nuclear export of incompletely spliced viral mRNAs.... more The Rev protein of HIV-1 is essential for the nuclear export of incompletely spliced viral mRNAs. This action depends on the mutationally defined Rev activation domain, which both binds the nucleoporin-like human cellular cofactor Rab/hRIP and also functions as a nuclear export signal. Protein kinase inhibitor alpha (PKI) also contains a potent nuclear export signal. However, PKI plays no role in nuclear RNA export and instead induces the nuclear export of a specific protein target, the catalytic subunit of cAMP-dependent protein kinase. Here, it is demonstrated that the nuclear export signal of PKI not only binds the Rab/hRIP cofactor specifically but also can effectively substitute for the Rev activation domain in mediating the nuclear export of HIV-1 mRNAs. We conclude that HIV-1 Rev and PKI act through an identical nuclear export pathway and that Rev, rather than using a dedicated RNA export pathway, is instead acting as an adaptor that allows viral mRNAs to access a cellular pr...

Research paper thumbnail of Distinct Functions of NS5A in Hepatitis C Virus RNA Replication Uncovered by Studies with the NS5A Inhibitor BMS-790052

Journal of Virology, 2011

BMS-790052, targeting nonstructural protein 5A (NS5A), is the most potent hepatitis C virus (HCV)... more BMS-790052, targeting nonstructural protein 5A (NS5A), is the most potent hepatitis C virus (HCV) inhibitor described to date. It is highly effective against genotype 1 replicons and also displays robust genotype 1 anti-HCV activity in the clinic (M. Gao et al., Nature 465:96-100, 2010). BMS-790052 inhibits genotype 2a JFH1 replicon cells and cell culture infectious virus with 50% effective concentrations (EC 50 s) of 46.8 and 16.1 pM, respectively. Resistance selection studies with the JFH1 replicon and virus systems identified drug-induced mutations within the N-terminal region of NS5A. F28S, L31M, C92R, and Y93H were the major resistance mutations identified; the impact of these mutations on inhibitor sensitivity between the replicon and virus was very similar. The C92R and Y93H mutations negatively impacted fitness of the JFH1 virus. Second-site replacements at NS5A residue 30 (K30E/Q) restored efficient replication of the C92R viral variant, thus demonstrating a genetic interac...

Research paper thumbnail of Intragenic Complementation of Hepatitis C Virus NS5A RNA Replication-Defective Alleles

Journal of Virology, 2012

Hepatitis C virus NS5A has three structural domains, is required for RNA replication and virion a... more Hepatitis C virus NS5A has three structural domains, is required for RNA replication and virion assembly, and exists in hypo- and hyperphosphorylated forms. Accumulated data suggest that phosphorylation is involved in modulating NS5A functions. We performed a mutational analysis of highly conserved serine residues in the linker region between domains I and II of genotype 2a JFH1 NS5A. As with genotype 1b Con1 NS5A, we found that specific serine residues were important for efficient hyperphosphorylation of JFH1 NS5A. However, in contrast with Con1 replicons, we observed a positive correlation between hyperphosphorylation and JFH1 replicon replication. We previously demonstrated trans -complementation of a hyperphosphorylation-deficient, replication-defective JFH1 replicon. Our results suggested that the defective NS5A encoded by this replicon, while lacking one NS5A function, was capable of performing a separate replication function. In this report, we examined an additional set of r...

Research paper thumbnail of 1424 Synergistic Interactions of HCV NS5A Replication Complex Inhibitors Sensitize Resistant Variants and Enhance the Efficacy of Daclatasvir (DCV, BMS-790052) in Vitro and in Vivo

Journal of Hepatology, 2013

LATE BREAKING ABSTRACTS 13.6±1.8 g/dL [8.8-17.5], respectively. After 12W, a complete early virol... more LATE BREAKING ABSTRACTS 13.6±1.8 g/dL [8.8-17.5], respectively. After 12W, a complete early virological response was obtained in 34 (83%) boceprevir patients and in 35 (61%) telaprevir patients (p = 0.026). Among 17 boceprevir and 16 telaprevir patients, 14 (82%) and 7 (43%) achieved an end of treatment response (EOT) with an undetetectable viral load, respectively (p = 0.032). Among 9 boceprevir and 5 telaprevir patients, 6 and 1 achieved SVR12, respectively. Among 6 patients in the boceprevir group, 3 achieved SVR24. In the telaprevir group, 29 patients discontinued therapy (serious adverse events, n = 13; virological breakthrough, n = 6; non-response, n = 9). In the boceprevir group, 14 patients discontinued therapy (serious adverse events, n = 5; virological breakthrough, n = 2; non-response, n = 4; retransplantation, n = 1). Four patients died in a context of infectious disorders: boceprevir, n = 2 (W20/W24); telaprevir, n = 2 (W2/W9). The most common side effect was anemia in 85% of patients: 95% and 96% in boceprevir and telaprevir groups received erythropoietin alone or combined with ribavirin dose reduction. Conclusion: In liver transplanted patients, EOT rate was 82% and 38% with boceprevir and telaprevir, respectively. Among the overall population, 44% of patients discontinued therapy because of treatment failure or occurrence of serious adverse events.