Biomedical subjects

M B Mathews

Publications and source records attributed to M B Mathews.

At least 19 recordsLinked to original sources

PubMed2001

Nuclear factor 90 is a substrate and regulator of the eukaryotic initiation factor 2 kinase double-stranded RNA-activated protein kinase.

Nuclear factor 90 (NF90) is a member of an expanding family of double-stranded (ds) RNA-binding proteins thought to be involved in gene expression. Originally identified in complex with nuclear factor 45 (NF45) as a sequence-specific DNA-binding protein, NF90 contains two double stranded RNA-binding motifs (dsRBMs) and interacts with highly structured RNAs as well as the dsRNA-activated protein kinase, PKR. In this report, we characterize the biochemical interactions between these two dsRBM containing proteins. NF90 binds to PKR through two independent mechanisms: an RNA-independent interaction occurs between the N terminus of NF90 and the C-terminal region of PKR, and an RNA-dependent interaction is mediated by the dsRBMs of the two proteins. Co-immunoprecipitation analysis demonstrates that NF90, NF45, and PKR form a complex in both nuclear and cytosolic extracts, and both proteins serve as substrates for PKR in vitro. NF90 is phosphorylated by PKR in its RNA-binding domain, and this reaction is partially blocked by the NF90 N-terminal region. The C-terminal region also inhibits PKR function, probably through competitive binding to dsRNA. A model for NF90-PKR interactions is proposed.

Animals↗

PubMed2001

Binding of double-stranded RNA to protein kinase PKR is required for dimerization and promotes critical autophosphorylation events in the activation loop.

Protein kinase PKR is activated by double-stranded RNA (dsRNA) and phosphorylates translation initiation factor 2alpha to inhibit protein synthesis in virus-infected mammalian cells. PKR contains two dsRNA binding motifs (DRBMs I and II) required for activation by dsRNA. There is strong evidence that PKR activation requires dimerization, but the role of dsRNA in dimer formation is controversial. By making alanine substitutions predicted to remove increasing numbers of side chain contacts between the DRBMs and dsRNA, we found that dimerization of full-length PKR in yeast was impaired by the minimal combinations of mutations required to impair dsRNA binding in vitro. Mutation of Ala-67 to Glu in DRBM-I, reported to abolish dimerization without affecting dsRNA binding, destroyed both activities in our assays. By contrast, deletion of a second dimerization region that overlaps the kinase domain had no effect on PKR dimerization in yeast. Human PKR contains at least 15 autophosphorylation sites, but only Thr-446 and Thr-451 in the activation loop were found here to be critical for kinase activity in yeast. Using an antibody specific for phosphorylated Thr-451, we showed that Thr-451 phosphorylation is stimulated by dsRNA binding. Our results provide strong evidence that dsRNA binding is required for dimerization of full-length PKR molecules in vivo, leading to autophosphorylation in the activation loop and stimulation of the eIF2alpha kinase function of PKR.

Amino Acid Sequence↗

PubMed2001

Three RNA polymerase II carboxyl-terminal domain kinases display distinct substrate preferences.

CDK7, CDK8, and CDK9 are cyclin-dependent kinases (CDKs) that phosphorylate the C-terminal domain (CTD) of RNA polymerase II. They have distinct functions in transcription. Because the three CDKs target only serine 5 in the heptad repeat of model CTD substrates containing various numbers of repeats, we tested the hypothesis that the kinases differ in their ability to phosphorylate CTD heptad arrays. Our data show that the kinases display different preferences for phosphorylating individual heptads in a synthetic CTD substrate containing three heptamer repeats and specific regions of the CTD in glutathione S-transferase fusion proteins. They also exhibit differences in their ability to phosphorylate a synthetic CTD peptide that contains Ser-2-PO(4). This phosphorylated peptide is a poor substrate for CDK9 complexes. CDK8 and CDK9 complexes, bound to viral activators E1A and Tat, respectively, target only serine 5 for phosphorylation in the CTD peptides, and binding to the viral activators does not change the substrate preference of these kinases. These results imply that the display of different CTD heptads during transcription, as well as their phosphorylation state, can affect their phosphorylation by the different transcription-associated CDKs.

Amino Acid Sequence↗

PubMed2001

Hepatitis C virus envelope protein E2 does not inhibit PKR by simple competition with autophosphorylation sites in the RNA-binding domain.

Double-stranded-RNA (dsRNA)-dependent protein kinase PKR is induced by interferon and activated upon autophosphorylation. We previously identified four autophosphorylated amino acids and elucidated their participation in PKR activation. Three of these sites are in the central region of the protein, and one is in the kinase domain. Here we describe the identification of four additional autophosphorylated amino acids in the spacer region that separates the two dsRNA-binding motifs in the RNA-binding domain. Eight amino acids, including these autophosphorylation sites, are duplicated in hepatitis C virus (HCV) envelope protein E2. This region of E2 is required for its inhibition of PKR although the mechanism of inhibition is not known. Replacement of all four of these residues in PKR with alanines did not dramatically affect kinase activity in vitro or in yeast Saccharomyces cerevisiae. However, when coupled with mutations of serine 242 and threonines 255 and 258 in the central region, these mutations increased PKR protein expression in mammalian cells, consistent with diminished kinase activity. A synthetic peptide corresponding to this region of PKR was phosphorylated in vitro by PKR, but phosphorylation was strongly inhibited after PKR was preincubated with HCV E2. Another synthetic peptide, corresponding to the central region of PKR and containing serine 242, was also phosphorylated by active PKR, but E2 did not inhibit this peptide as efficiently. Neither of the PKR peptides was able to disrupt the HCV E2-PKR interaction. Taken together, these results show that PKR is autophosphorylated on serine 83 and threonines 88, 89, and 90, that this autophosphorylation may enhance kinase activation, and that the inhibition of PKR by HCV E2 is not solely due to duplication of and competition with these autophosphorylation sites.

Amino Acid Sequence↗

PubMed2000

Functional characterization of and cooperation between the double-stranded RNA-binding motifs of the protein kinase PKR.

The interferon-inducible double-stranded RNA (dsRNA)-activated protein kinase PKR is regulated by dsRNAs that interact with the two dsRNA-binding motifs (dsRBMs) in its N terminus. The dsRBM is a conserved protein motif found in many proteins from most organisms. In this study, we investigated the biochemical functions and cytological activities of the two PKR dsRBMs (dsRBM1 and dsRBM2) and the cooperation between them. We found that dsRBM1 has a higher affinity for binding to dsRNA than dsRBM2. In addition, dsRBM1 has RNA-annealing activity that is not displayed by dsRBM2. Both dsRBMs have an intrinsic ability to dimerize (dsRBM2) or multimerize (dsRBM1). Binding to dsRNA inhibits oligomerization of dsRBM1 but not dsRBM2 and strongly inhibits the dimerization of the intact PKR N terminus (p20) containing both dsRBMs. dsRBM1, like p20, activates reporter gene expression in transfection assays, and it plays a determinative role in localizing PKR to the nucleolus and cytoplasm of the cell. Thus, dsRBM2 has weak or no activity in dsRNA binding, stimulation of gene expression, and PKR localization, but it strongly enhances these functions of dsRBM1 when contained in p20. However, dsRBM2 does not enhance the annealing activity of dsRBM1. This study shows that the dsRBMs of PKR possess distinct properties and that some, but not all, of the functions of the enzyme depend on cooperation between the two motifs.

Amino Acid Motifs↗

PubMed2000

Human breast cancer cells contain elevated levels and activity of the protein kinase, PKR.

PKR is a double-stranded (ds) RNA activated protein kinase whose expression is induced by interferon. Activated PKR phosphorylates its cellular substrate, eIF2, an essential initiation factor of translation. Prior evidence from a murine model system suggested that PKR may act as a tumor suppressor, but the evidence from human tumors is equivocal. To study PKR function in human breast cancer, PKR activity was measured in mammary carcinoma cell lines and nontransformed mammary epithelial cell lines. If PKR functioned as a tumor suppressor in this system, its activity would be higher in nontransformed cells than in carcinoma cells. On the contrary, PKR autophosphorylation and the phosphorylation of its substrate, the alpha-subunit of eIF2, is 7 - 40-fold higher in lysates prepared from breast carcinoma cell lines than in those from nontransformed epithelial cell lines. Correspondingly, a larger proportion of eIF2alpha is present in a phosphorylated state in carcinoma cell lines than in nontransformed cell lines. Protein synthesis is not inhibited by the high eIF2alpha phosphorylation in carcinoma cells, probably because they contain higher levels of eIF2B, the initiation factor that is inhibited by eIF2alpha phosphorylation. The dramatically lower PKR activity in nontransformed cell lines is partially due to lower PKR protein levels (2 - 4-fold) as well as to the presence of a PKR inhibitor. The nontransformed cells contain P58, a known cellular inhibitor of PKR that physically interacts with PKR and may be responsible for the low PKR activity in these cells. Taken together, these observations call into question the role of PKR as a tumor suppressor and suggest a positive regulatory role of PKR in growth control of breast cancer cells.

Blotting, Western↗

PubMed2000

Proteins binding to duplexed RNA: one motif, multiple functions.

Highly structured and double-stranded (ds) RNAs are adaptable and potent biochemical entities. They interact with dsRNA-binding proteins (RBPs), the great majority of which contain a sequence called the dsRNA-binding motif (dsRBM). This approximately 70-amino-acid sequence motif forms a tertiary structure that interacts with dsRNA, with partially duplexed RNA and, in some cases, with RNA-DNA hybrids, generally without obvious RNA sequence specificity. At least nine families of functionally diverse proteins contain one or more dsRBMs. The motif also participates in complex formation through protein-protein interactions.

Amino Acid Motifs↗

PubMed2000

Expanded CUG repeat RNAs form hairpins that activate the double-stranded RNA-dependent protein kinase PKR.

Myotonic dystrophy is caused by an expanded CTG repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene. The expanded repeat triggers the nuclear retention of mutant DMPK transcripts, but the resulting underexpression of DMPK probably does not fully account for the severe phenotype. One proposed disease mechanism is that nuclear accumulation of expanded CUG repeats may interfere with nuclear function. Here we show by thermal melting and nuclease digestion studies that CUG repeats form highly stable hairpins. Furthermore, CUG repeats bind to the dsRNA-binding domain of PKR, the dsRNA-activated protein kinase. The threshold for binding to PKR is approximately 15 CUG repeats, and the affinity increases with longer repeat lengths. Finally, CUG repeats that are pathologically expanded can activate PKR in vitro. These results raise the possibility that the disease mechanism could be, in part, a gain of function by mutant DMPK transcripts that involves sequestration or activation of dsRNA binding proteins.

Base Pairing↗

PubMed1999

Dual action of the adenovirus E1A 243R oncoprotein on the human proliferating cell nuclear antigen promoter: repression of transcriptional activation by p53.

The promoter of the human proliferating cell nuclear antigen (PCNA) gene is activated by the adenovirus oncoprotein E1A 243R in HeLa cells. To understand the effect of this oncoprotein on PCNA expression in cells that are sensitive to oncogenic transformation by adenovirus, we studied the effect of E1A 243R on PCNA promoter-directed reporter gene expression in cloned rat embryo fibroblast (CREF) and primary baby rat kidney cells. In contrast to the results obtained in HeLa cells, E1A repressed the PCNA promoter in both cell-types. Promoter analysis identified a p53-responsive element that mediates E1A-induced repression. Repression required the intact N-terminus of E1A 243R, as shown by the ability of mutant E1A proteins to repress the promoter, and correlated with the p300-binding region of E1A. The adenovirus E1B 19K protein relieved repression by E1A 243R. These results reveal dual pathways for induction of this essential DNA replication factor and suggest a mechanism for oncogenic cooperativity between the E1A and E1B oncoproteins.

Adenovirus E1A Proteins↗

PubMed1999

Involvement of RFX1 protein in the regulation of the human proliferating cell nuclear antigen promoter.

The proliferating cell nuclear antigen (PCNA) is an essential eukaryotic DNA replication factor that is transcriptionally regulated by the adenovirus oncoprotein E1A 243R. Inducibility of the human PCNA promoter by E1A 243R is conferred by the cis-acting PCNA E1A-responsive element (PERE), which associates with the ATF-1, cAMP response element-binding protein (CREB), and RFX1 transcription factors and is modulated by cellular proteins such as the coactivator CREB-binding protein (CBP) and tumor suppressor p107 (Labrie, C., Lee, B. H., and Mathews, M. B. (1995) Nucleic Acids Res. 23, 3732-3741; Lee, B. H., and Mathews, M. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4481-4486; Lee, B. H., Liu, M., and Mathews, M. B. (1998) J. Virol. 72, 1138-1145). RFX1 also forms a complex with sequences in the PCNA promoter of mouse and rat that share homology with the RFX1 consensus site. To explore the role of RFX1 in regulating the PCNA promoter, we examined the effects of mutations in the human PERE on RFX1 binding and gene expression. Mutations within the RFX1 consensus binding site reduced RFX1 binding, whereas mutations upstream of the site, or on its border, increased RFX1 binding. These mutations also affected the transcriptional activity of PCNA-chloramphenicol acetyltransferase reporter constructs in transient expression assays. The relative transcriptional activity of mutant PCNA promoters, both in the presence and absence of E1A 243R, was inversely related to their ability to complex with RFX1. These findings suggest that the binding of RFX1 is influenced by sequences outside its consensus binding site and that this transcription factor plays an inhibitory role in the regulation of PCNA gene expression.

Adenovirus E1A Proteins↗

PubMed1999

Termination sequence requirements vary among genes transcribed by RNA polymerase III.

RNA polymerase III (pol III) transcription generally terminates at a run of four or more thymidine (T) residues but some pol III genes contain runs of T residues that are not recognized as termination signals. Here, we investigate the terminal signal requirements that are operative in adenovirus virus-associated (VA) RNA genes. In the Xenopus 5 S RNA gene, efficient termination requires the T residues to be in a G+C-rich sequence context, but a run of five T residues in a G+C-rich context does not cause pol III termination when placed 30 nt downstream of the adenovirus-2 VA RNAI promoter in a VA-Tat chimeric gene. The failure of pol III to recognize this putative termination signal is not due to the chimeric nature of the gene or to the proximity of the signal to the promoter, but to its sequence context. Termination at the VA RNA gene site requires a T-rich sequence and is inhibited by the proximity of G residues, but is insensitive to the presence of A residues. The T-rich sequence need not be uninterrupted, however. In the VA RNA gene of the avian adenovirus, CELO, the first of two tandem termination signals contains an interrupted run of T residues, TTATT, which functions as a terminator with high (although not complete) efficiency. These findings, together with a survey of sequences neighboring the terminal site of other pol III genes, lead to the conclusion that pol III termination signals are more complex than hitherto recognized, and that sequence context requirements differ between members of the class 1 and class 2 families of pol III genes.

Animals↗

PubMed1999

Inosine and N1-methylinosine within a synthetic oligomer mimicking the anticodon loop of human tRNA(Ala) are major epitopes for anti-PL-12 myositis autoantibodies.

Sera of some patients afflicted with the inflammatory disease myositis contain antibodies of the anti-PL-12 type. A fraction of these polyclonal autoantibodies specifically precipitates the fully matured human tRNA(Ala) bearing the anticodon IGC (PL-12 antigen). Earlier work (Bunn & Mathews, 1987, Science 238:116-119) had shown that the epitopes are located entirely within the anticodon stem-loop of the tRNA(Ala). Here we demonstrate that human anti-tRNA(Ala) autoantibodies immunoprecipitate a synthetic polyribonucleotide containing inosine (I) and N1-methylinosine (m1I) separated by 2 nt as in the anticodon stem-loop of human tRNA(Ala). The shortest polyribonucleotide that can be immunoprecipitated corresponds to the pentanucleotide IpGpCpm1IpUp, which corresponds to part of the anticodon loop of human tRNA(Ala) and lacks the stem-loop structure. The efficiency of immunoprecipitation was about four times greater with longer polyribonucleotides capable of forming a stem-loop structure, and was abolished by altering the relative positions of I and m1I within the synthetic polynucleotide. Synthetic oligodeoxyribonucleotide analogs of the tRNA(Ala) stem-loop, containing the sequence dIpdGdCdm1Ip, are not antigenic. Our results show that human anti-tRNA(Ala) autoantibodies selectively recognize chemical details of modified nucleotides (the 6-keto group of inosine-34 and the 6-keto group and the N1-methyl groups of N1-methylinosine-37) within an anticodon loop structure of a tRNA molecule. We also describe the chemical synthesis of the phosphoramidite derivatives corresponding to N1-methylinosine and N1-methyl-2'-deoxyinosine for use in the automatic chemical synthesis of oligonucleotides containing N1-methylinosine and N1-methyl-2'-deoxyinosine.

Antibodies, Antiphospholipid↗

PubMed1999

Human and rodent transcription elongation factor P-TEFb: interactions with human immunodeficiency virus type 1 tat and carboxy-terminal domain substrate.

The human immunodeficiency virus type 1 transcriptional regulator Tat increases the efficiency of elongation, and complexes containing the cellular kinase CDK9 have been implicated in this process. CDK9 is part of the Tat-associated kinase TAK and of the elongation factor P-TEFb (positive transcription elongation factor-b), which consists minimally of CDK9 and cyclin T. TAK and P-TEFb are both able to phosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II, but their relationships to one another and to the stimulation of elongation by Tat are not well characterized. Here we demonstrate that human cyclin T1 (but not cyclin T2) interacts with the activation domain of Tat and is a component of TAK as well as of P-TEFb. Rodent (mouse and Chinese hamster) cyclin T1 is defective in Tat binding and transactivation, but hamster CDK9 interacts with human cyclin T1 to give active TAK in hybrid cells containing human chromosome 12. Although TAK is phosphorylated on both serine and threonine residues, it specifically phosphorylates serine 5 in the CTD heptamer. TAK is found in the nuclear and cytoplasmic fractions of human cells as a large complex (approximately 950 kDa). Magnesium or zinc ions are required for the association of Tat with the kinase. We suggest a model in which Tat first interacts with P-TEFb to form the TAK complex that engages with TAR RNA and the elongating transcription complex, resulting in hyperphosphorylation of the CTD on serine 5 residues.

Amino Acid Sequence↗

PubMed1998

Activities of adenovirus virus-associated RNAs: purification and characterization of RNA binding proteins.

Most human adenoviruses encode two virus-associated (VA) RNAs, VA RNAI and VA RNAII, that accumulate to high levels in the cytoplasm of infected cells. The function of VA RNAI in blocking the activation of the cellular kinase PKR is well known, but the role of VA RNAII is obscure. Herein we characterize and purify several human proteins that interact preferentially with VA RNAII in Northwestern blot assays. Two of these proteins were identified as RNA helicase A and NF90, a component of the heterodimeric nuclear factor of activated T cells (NFAT). They copurified with the smaller NFAT subunit, NF45, which did not bind VA RNAII, and with an unidentified protein, p97, which did bind VA RNAII. Both RNA helicase A and NF90 contain two copies of a double-stranded (ds) RNA binding motif and bind strongly to dsRNA. NF90 interacts with RNAs in the following order of affinity: dsRNA > VA RNAII > VA RNAI > single-stranded RNA. Furthermore, VA RNAII is more effective than VA RNAI as an inhibitor of RNA helicase activity. These data identify RNA helicase A and NF90 as cellular proteins with an affinity for dsRNA and other structured RNA molecules and suggest that their functions are subject to regulation by RNA ligands including VA RNAII.

Adenoviridae↗

PubMed1998

RNA binding and modulation of PKR activity.

PKR is an RNA-dependent protein kinase that is induced in mammalian cells by interferon treatment. It is present in a latent or inactive form in mammalian cells and is activated by very low concentrations of double-stranded (ds) RNA. Activated PKR phosphorylates eIF2, an essential initiation factor of protein synthesis, as well as other substrates including histone IIA, a 90-kDa protein from rabbit reticulocytes, the inhibitor, IkappaB, of the transcription factor, NF-kappaB, and the HIV-1 Tat protein. PKR interacts with several cellular and viral products and these interactions modulate its activation by dsRNA. Here we describe methods that are used to study the activation or inhibition of PKR by RNA modulators. Specifically, we detail (1) the purification of PKR from interferon-treated mammalian cells, (2) functional assays for PKR activation and inhibition in vitro, using purified enzyme or crude cell lysates, and (3) assays allowing evaluation of the binding of dsRNA and single-stranded RNA to PKR.

Chromatography, Liquid↗

PubMed1998

Regulation of the human proliferating cell nuclear antigen promoter by the adenovirus E1A-associated protein p107.

The adenovirus E1A 243R oncoprotein is capable of transactivating the expression of the human proliferating cell nuclear antigen (PCNA) promoter. Mutational analysis of the E1A 243R protein suggested that both its p300/CBP- and p107-binding regions are required for optimal induction of the PCNA promoter (C. Kannabiran, G. F. Morris, C. Labrie, and M. B. Mathews, J. Virol. 67:425-437, 1993). We show that overexpression of p107 antagonizes the induction of PCNA by E1A 243R in transient expression assays. This inhibition is largely independent of p107's ability to interact with E1A 243R, because p107 mutants unable to bind to E1A 243R retain the ability to repress the E1A-activated PCNA promoter. Electrophoretic mobility shift assays with the PCNA promoter detected the presence of p107 in one of the major DNA-protein complexes, EH1, formed with HeLa cell nuclear extracts. Promoter mutations that disrupt the formation of complex EH1 abrogated p107's ability to reverse E1A 243R-induced PCNA expression. The same mutations characterize a sequence important for the binding of transcription factor RFX1 (C. Labrie, G. F. Morris, and M. B. Mathews, Nucleic Acids Res. 23:3732-3741, 1995), implying that p107 antagonizes E1A 243R-induced PCNA expression through this RFX1-binding site. Our data are suggestive of a novel cooperative mechanism for transactivation of PCNA expression, in which E1A 243R relieves transcriptional repression exerted by p107 on the promoter.

Adenoviridae↗

PubMed1998

Autophosphorylation in the activation loop is required for full kinase activity in vivo of human and yeast eukaryotic initiation factor 2alpha kinases PKR and GCN2.

The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit alpha (eIF2alpha) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop, described previously for MAP kinase, is a regulatory feature conserved in PKR. We found that the yeast eIF2alpha kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR. Thr-887 was more critically required than was Thr-882 for GCN2 kinase activity, paralleling the relative importance of Thr-446 and Thr-451 in PKR. These results indicate striking similarities between GCN2 and PKR in the importance of autophosphorylation and the conserved Thr residues in the activation loop.

3T3 Cells↗

PubMed1997

Effects of heterologous downstream sequences on the activity of the HIV-1 promoter and its response to Tat.

In HIV-1 infection, Tat acts at least in part to control transcriptional elongation by overcoming premature transcriptional termination. In some other genes this process is governed by DNA elements called attenuators in concert with cellular transcription factors. To understand the action of Tat more fully and explore its role as an anti-attenuator, we examined the ability of several natural and synthetic attenuation sequences to modulate transcription initiated at the HIV LTR. Fragments containing these signals were inserted downstream of the TAR element in an HIV-CAT chimera and their effects on transcription were assessed both in vitro and in vivo. Runoff transcription assays in HeLa cell extracts demonstrated that the attenuators give rise to premature termination of transcripts initiated from the heterologous HIV-LTR promoter in vitro. When transiently expressed following transfection into Cos cells, however, premature transcript termination at the attenuation site was not observed. Nevertheless, many of the inserted sequences exerted marked effects on CAT gene expression and on transactivation by Tat at both the RNA and protein levels. The nature and magnitude of the effects depended upon the identity of the attenuator and its orientation but only one of 16 sequences tested met the criteria for a Tat-suppressible attenuator in vivo. One other sequence, in contrast, severely reduced Tat-activated transcription without inhibiting basal transcription These results indicate that sequences downstream of the HIV LTR can influence its function as a promoter and its response to Tat transactivation, but lend little support to their role as attenuators in vivo.

Animals↗