Roles of the U5 snRNP in spliceosome dynamics and catalysis.

Most protein-coding genes in eukaryotes are interrupted by non-coding intervening sequences (introns), which must be precisely removed from primary gene transcripts (pre-mRNAs) before translation of the message into protein. Intron removal by pre-mRNA splicing occurs in the nucleus and is catalysed by complex ribonucleoprotein machines called spliceosomes. These molecular machines consist of several small nuclear RNA molecules and their associated proteins [together termed snRNP (small nuclear ribonucleoprotein) particles], plus multiple accessory factors. Of particular interest are the U2, U5 and U6 snRNPs, which play crucial roles in the catalytic steps of splicing. In the present review, we summarize our current understanding of the role played by the protein components of the U5 snRNP in pre-mRNA splicing, which include some of the largest and most highly conserved nuclear proteins.

Synthesis and RNA polymerase incorporation of the degenerate ribonucleotide analogue rPTP.

The synthesis and enzymatic incorporation into RNA of the hydrogen bond degenerate nucleoside analogue 6-(beta-d-ribofuranosyl)-3, 4-dihydro-8H-pyrimido[4,5-c]-[1,2]oxazin-7-one (P) is described. The 5'-triphosphate of this analogue is readily incorporated by T3, T7 and SP6 RNA polymerases into RNA transcripts, being best incorporated in place of UTP, but also in place of CTP. When all the uridine residues in an HIV-1 TAR RNA transcript are replaced by P the transcript has similar characteristics to the wild-type TAR RNA, as demonstrated by similar melting temperatures and CD spectra. The P-substituted TAR transcript binds to the Tat peptide ADP-1 with only 4-fold lowered efficiency compared with wild-type TAR.

Transfer of Tat and release of TAR RNA during the activation of the human immunodeficiency virus type-1 transcription elongation complex.

The HIV-1 trans-activator protein, Tat, is a potent activator of transcriptional elongation. Tat is recruited to the elongating RNA polymerase during its transit through the trans-activation response region (TAR) because of its ability to bind directly to TAR RNA expressed on the nascent RNA chain. We have shown that transcription complexes that have acquired Tat produce 3-fold more full-length transcripts than complexes not exposed to Tat. Western blotting experiments demonstrated that Tat is tightly associated with the paused polymerases. To determine whether TAR RNA also becomes attached to the transcription complex, DNA oligonucleotides were annealed to the nascent chains on the arrested complexes and the RNA was cleaved by RNase H. After cleavage, the 5' end of the nascent chain, carrying TAR RNA, is quantitatively removed, but the 3' end of the transcript remains associated with the transcription complex. Even after the removal of TAR RNA, transcription complexes that have been activated by Tat show enhanced processivity. We conclude that Tat, together with cellular co-factors, becomes attached to the transcription complex and stimulates processivity, whereas TAR RNA does not play a direct role in the activation of elongation and is used simply to recruit Tat and cellular co-factors.

The human immunodeficiency virus long terminal repeat includes a specialised initiator element which is required for Tat-responsive transcription.

The effects of mutations in human immunodeficiency virus type-1 (HIV-1) long terminal repeat on initiation and on Tat-mediated trans-activation were studied using cell-free transcription assays. All the elements that are necessary for efficient transcription initiation in vitro are included in the core promoter. This region contains three tandem Sp1 binding sites, a TATA element and an initiator (INR) sequence. Although the HIV-1 INR element overlaps the trans-activation response region (TAR), it forms an integral part of the promoter. The HIV-1 INR element was characterised in detail using a template that carries a complete HIV-1 promoter and a displaced TAR RNA element. The results demonstrate that the sequence G+1GGTCT is essential for HIV-1 INR function. RNase protection experiments show that Tat acts exclusively to stimulate transcriptional elongation. Mutations in the core promoter elements reduce initiation rates dramatically but do not block Tat activity. For each mutation studied, the total level of transcription in the presence of Tat is proportional to the rate of initiation in the absence of Tat. Furthermore the rate of initiation remains constant in the presence or absence of Tat. We conclude that the elements of the HIV-1 core promoter act in concert to simulate initiation. By contrast, Tat acts independently of the core promoter elements and stimulates elongation. The data strongly suggest that Tat is recruited to the elongating transcription complex during its transit through TAR.

The RNA element encoded by the trans-activation-responsive region of human immunodeficiency virus type 1 is functional when displaced downstream of the start of transcription.

The human immunodeficiency virus type 1 (HIV-1) trans-activator protein, Tat, specifically stimulates transcription from the viral long terminal repeat. Tat binds to an RNA stem-loop structure encoded by the trans-activation response region (TAR). To test whether TAR is functional when displaced downstream of the start of transcription, we assayed a series of templates carrying duplicated TAR elements in cell-free transcription systems. When the normally positioned TAR element (TAR-1) is inactivated by mutations in either the Tat binding site or the apical loop sequence, which acts as the binding site for a cellular factor, transactivation can be rescued by a wild-type TAR element placed downstream (TAR-2). The TAR-2 element is functional even when placed > 200 nt downstream of TAR-1. TAR complementation experiments have also shown that a functional TAR element requires both an intact Tat binding site and an intact apical loop sequence. For example, if TAR-1 carries a mutation in the loop element it cannot be rescued by a TAR-2 element carrying a mutation in the Tat binding site. Substitution mutations in TAR-1 show that the 5' half of TAR also encodes an essential DNA element which is required for efficient transcription initiation. These results strongly suggest that Tat and cellular cofactors which bind TAR RNA associate with the transcription complex during its transit through TAR.

Human immunodeficiency virus type 1 transactivator protein, tat, stimulates transcriptional read-through of distal terminator sequences in vitro.

The human immunodeficiency virus type 1 transactivator protein, tat, specifically stimulates transcription from the viral long terminal repeat. We used cell-free transcription systems to test whether tat can stimulate transcriptional read-through of an artificial terminator sequence (e.g., a stable RNA stem-loop structure followed by a tract of nine uridine residues) placed downstream of the viral long terminal repeat. In the absence of tat, RNA polymerases are prematurely released from the template at the terminator sequence. Recombinant tat protein purified from Escherichia coli increased the synthesis of full-length transcripts approximately 25-fold and decreased the amount of transcripts ending at the terminator sequence. The reaction is strictly dependent upon the presence of a functional transactivation-responsive region (TAR) sequence. Mutations in the tat binding site on TAR RNA and mutations in the TAR RNA loop block transactivation in vivo. Neither type of mutation is able to respond to tat in vitro. These results strongly suggest that after transcription through the TAR region, tat modifies the transcription complex to increase its elongation capacity.

High affinity binding of TAR RNA by the human immunodeficiency virus type-1 tat protein requires base-pairs in the RNA stem and amino acid residues flanking the basic region.

The binding site for tat protein on TAR RNA has been defined in quantitative terms using an extensive series of mutations. The relative dissociation constants for the mutant TAR RNAs were measured using a dual-label competition filter binding assay in which 35S-labelled wild-type TAR RNA (K1) was competed against 3H-labelled mutant TAR RNA (K2). The error in the self-competition experiment was usually less than 10% (e.g. K2/K1 = 1.07 +/- 0.05, n = 19) and the experimental data accurately matched theoretical curves calculated with fitted dissociation constants. Mutations in U23, a critical residue in the U-rich "bulge" sequence, or in either of the two base-pairs immediately above the "bulge", G26.C39 and A27.U38 reduced that affinity by 8- to 20-fold. Significant contributions to tat binding affinity were also made by the base-pairs located immediately below the bulge. For example, mutation of A22.U40 to U.A reduced tat affinity 5-fold, and mutation of G21.C41 to C.G reduced tat affinity 4-fold. The binding of a series of peptides spanning the basic "arginine-rich" sequence of tat was examined using both filter-binding and gel mobility shift assays. Each of the peptides showed significantly reduced affinities for wild-type TAR RNA compared to the tat protein. The ADP-2 (residues 43 to 72), ADP-3 (residues 48 to 72) and ADP-5 (residues 49 to 86) peptides were unable to discriminate between wild-type TAR RNA and TAR RNA mutants with the same fidelity as the tat protein. For example, these peptides showed no more than 3-fold reductions in affinity relative to wild-type TAR RNA for the U23-->C mutation in the bulge, or G26.G39-->C.G mutation in the stem of TAR RNA. By contrast, the ADP-I (residues 37 to 72), ADP-4 (residues 32 to 62) and ADP-6 (residues 32 to 72) peptides, which each carry amino acid residues from the "core" region of the tat protein have binding specificities that more closely resemble the protein. The ADP-4 and ADP-6 peptides showed between 4- and 7-fold reductions in affinity for the U23-->C or G26.C39-->C.G mutations. The ADP-1 peptide most closely resembles the protein in its binding specificity and showed 9-fold and 14-fold reductions in affinity for the two mutants, respectively. Chemical-modification interference assays using diethylpyrocarbonate (DEPC) and ethylnitrosourea (ENU) were also used to compare the binding properties of the tat protein and the tat-derived peptides.(ABSTRACT TRUNCATED AT 400 WORDS)

The region of the envelope gene of human immunodeficiency virus type 1 responsible for determination of cell tropism.

Different isolates of human immunodeficiency virus type 1 (HIV-1) vary in the cell tropisms they display, i.e., the range of cell types in which they are able to establish a productive infection. Here, we report on the phenotypes of recombinants between two molecularly cloned strains of HIV-1. Our results prove that the envelope glycoprotein gp120 is solely responsible for the difference in cell tropism between the two parental isolates and that no other genes or sequences are involved in determining the cell tropism of these strains. The region of the envelope involved in the determination of cell tropism includes sequences which encode the V3 loop of gp120. Control of cell tropism by this region of the virus env gene is a general phenomenon which applies to many different HIV-1 isolates.

Production and characterisation of a monoclonal antibody to human papillomavirus type 16 using recombinant vaccinia virus.

A monoclonal antibody was raised against the major capsid protein L1 of human papillomavirus type 16, using a recombinant vaccinia virus that expresses the L1 protein, as a target for screening. This antibody, designated CAMVIR-1, reacted with a 56 kilodalton protein in cells infected with L1-vaccinia virus, and the protein was present in a predominantly nuclear location. The antibody also detects the HPV-16 L1 antigen in formalin fixed, paraffin wax embedded biopsy specimens and on routine cervical smears. The antibody reacts strongly and consistently with biopsy specimens containing HPV-16 or HPV-33, but very weak reactions were occasionally observed with biopsy specimens or smears containing HPV-6 or HPV-11. The potential advantages of using a vaccinia recombinant are (i) the target protein is synthesised in a eukoryotic cell so that its "processing" and location are normal; (ii) cells infected with vaccinia recombinants can be subjected to various fixing procedures similar to those used for routine clinical material. This greatly increases the probability that an identified antibody will be useful in a clinical setting.

Analysis of the L1 gene product of human papillomavirus type 16 by expression in a vaccinia virus recombinant.

The L1 open reading frame of human papillomavirus type 16 (HPV16) has been expressed in vaccinia virus under the control of both the 7.5K early and late promoter, and the 4b major late promoter. Antibodies to a beta-galactosidase fusion protein containing a C-terminal portion of the HPV16 L1 gene product were used to compare the levels of L1 expression in the two recombinants, and showed that greater levels of expression were obtained when the gene was placed under the control of the 4b late promoter. Immunofluorescence studies revealed a nuclear location of the L1 gene product when expressed in vaccinia virus. Antibodies to the beta-galactosidase fusion protein detected a major polypeptide species of 57K and a minor species of 64K in Western blots of recombinant-infected cell lysates. The 64K species was not detected when cells were infected in the presence of tunicamycin, indicating that the primary translation product of the HPV16 L1 open reading frame is modified by N-linked glycosylation when expressed in vaccinia virus. Whereas antibodies to HPV16 L1 fusion proteins and to a peptide containing amino acids from the C terminus of HPV16 L1 reacted well in Western blots with the HPV16 L1 target expressed in vaccinia virus, no reactivity was observed with antibodies to bovine papillomavirus type 1 particles or to a HPV6b fusion protein.

Cooperative effects between two acyclovir resistance loci in herpes simplex virus.

The acyclovir-resistant mutant SC16 R9C2 (H.J. Field, G. Darby, and P. Wildy , J. Gen. Virol. 49:115-124, 1980) has been shown to contain two resistance loci which segregate independently on recombination with wild-type virus. One locus is in thymidine kinase, and the other is in DNA polymerase. Both induced enzymes have altered properties, thymidine kinase showing a low affinity for acyclovir and low activity, and DNA polymerase showing a low affinity for acyclovir triphosphate. Other properties of both enzymes are described which distinguish them from their wild-type counterparts. Recombinants containing either mutant thymidine kinase ( RSC -11) or mutant DNA polymerase ( RSC -26), but not both, have been used to investigate the relative contribution of each lesion to resistance and pathogenicity. Although SC16 R9C2 and both recombinants grow as well as does wild-type virus in tissue culture, they are considerably attenuated in vivo, the greatest attenuation of virulence being seen with SC16 R9C2 and RSC -26. With respect to both acyclovir resistance and in vivo growth, the lesions appear to behave synergistically. Cross resistance studies have shown the recombinant RSC -26, which contains mutant DNA polymerase but which evidently expresses wild-type thymidine kinase, to be cross resistant to both 5-iodo-2'-deoxyuridine and 5-trifluoromethyl-2'-deoxyuridine but not to (E)-5-(2-bromovinyl)-2'-deoxyuridine or 9-beta-D-arabinofuranosyladenine.