Accumulation of bacteriophage T7 head-related particles in an Escherichia coli mutant.

We have analyzed the structure of the late cytoplasmic RNAs made after infection with wild-type simian virus 40 and a set of viable mutants, four of which have deletions and one an insertion within the nucleotide sequence specifying the leader segment of the 16S and 19S mRNA's. The principal findings are: (i) simian virus 40 16S and 19S mRNA's made during infections with wild-type virnds and possibly in the nucleotide sequence comprising the "leader" segments. (II) "Spliced" 16S and 19S mRNA's are made during infections with each of the mutants although, in some cases, the ratio of 19S to 16S mRNA species is reduced. (iii) The deletion or insertion of nucleotides within the DNA segment defined by map position 0.70 to 0.75 causes striking alterations in the types of leader structures in the late mRNAs. (iv) Many of the late RNA leader segments produced after infection with the mutants appear to be multiply spliced, i.e., instead of the major 200- to 205-nucleotide-long leader segment present in wild-type 16S mRNA, the RNAs produced by several of the deletion mutants have leaders with whort discontiguous segments.     

Expression of adenovirus type 12 E1A gene in monkey cells, using a simian virus 40 vector.

Simian virus 40 (SV40) recombinants carrying the adenovirus type 12 E1A gene were constructed. The SV40 expression vector was constructed by removing most of the VP1 gene and an internal part of the intervening sequence for late 16S RNA and by joining the 5' and 3' splice sites into a small segment. The adenovirus type 12 E1A gene with or without its own promoter was inserted downstream from the SV40 late promoter and the splicing junctions. The recombinant DNA was propagated and packaged in monkey cells by cotransfection with an early temperature-sensitive mutant (tsA58) DNA as helper. Immunofluorescent staining of the monkey cells infected with the resulting virus stocks showed that up to 20% of the cells overproduced the E1A gene products in the nuclei. Two-dimensional gel electrophoresis of the products indicated that the products were very similar or identical to the authentic polypeptides synthesized in adenovirus type 12-infected human embryo kidney cells. The E1A mRNA was initiated at the SV40 late promoter irrespective of the presence of the E1A promoter and terminated at either the E1A or the SV40 polyadenylation signal. These hybrid mRNAs were correctly spliced in the E1A coding region.     

Ability of nonpermissive mouse cells to express a simian virus 40 late function(s)

Mouse cells are fully nonpermissive for simian virus 40 (SV40). Infection does not lead to detectable virus replication. In this report, it was shown, first, that spliced 16S and 19S SV40 late mRNA were present in cytoplasmic and polysomal polyadenylated acid+ RNA preparations from SV40-infected baby mouse kidney cells. The 16S and 19S SV40 late mRNA's produced in infected baby mouse kidney cells were identical to or similar to the 16S and 19S SV40 late mRNA's produced in permissive monkey cells as judged by their S1 mapping patterns performed with the late strand of HpaII-BamHI fragment B and by their sedimentation patterns in a sucrose gradient. It was also shown that the 16S late mRNA from infected baby mouse kidney cells could be translated into a polypeptide which was identical to or similar to virion protein VP1 in every aspect examined, including the patter of peptide mapping by limited proteolysis. Second, we reported that mouse kidney cells produced detectable, although low, levels of SV40 virion protein VP1, as shown by the sodium dodecyl sulfate-polyacrylamide gel autoradiogram of [35S]methionine-labeled proteins immunoprecipitated by a rabbit antiserum directed against SV40 virion proteins. Third, it was reported that infected baby mouse kidney cells produced late mRNA's either (i) when the infection was done at a restrictive temperature with the nonleaky tsA58 mutant or (ii) in cells treated with 100 microgram of cycloheximide per ml, in which large T antigen synthesis was inhibited by more than 99.9%. This suggested that large T antigen was not required for the synthesis of late mRNA in mouse cells.     

Activation of a cryptic splice donor in human immunodeficiency virus type-1

The human immunodeficiency virus type-1 regulatory protein Rev is absolutely required for the production of viral structural proteins. Splice sites have been seen to function ascis-acting repressor sequendes (CRS) and inhibit expression of the Rev-dependent RNAs. In order to analyze the role of a splice donor in Rev dependence, the wild-type 5 splice donor of HIV-1 was mutated in the context of othergag sequences. Following transient transfection, RNA expression by RT-PCR was analyzed. The unspliced RNA produced by the mutant construct still required Rev for the cytoplasmic accumulation of the RNA. Despite deletion of the wild-type 5 splice donor and thetat splice acceptor was used. A cryptic splice donor was identified by PCR and subsequent cloning of the spliced RNA. The cryptic site is 5/9 to the consensus sequence and located immediately downstream of the initiation codon (ATG) for Gag. Analysis of the RNA product containing the cryptic splice donor revealed that the Rev was required for the cytoplasmic accumulation of unspliced RNA, while spliced RNA was Rev independent. Transfection of a wild-type construct also demonstrated usage of the cryptic splice donor. These results indicate that a cryptic splice donor can be activated when the wild-type splice donor is inactivated and that the cryptic splice donor may retain Rev regulation. The findings also suggest the potential for cryptic splice sites to serve as CRS in the determining the Rev dependence of viral RNAs.     

Altered utilization of splice sites and 5'' termini in late RNAs produced by leader region mutants of simian virus 40.

We compared the 5' termini and splices of the late 16S and 19S RNAs synthesized by wild-type simian virus 40 and five mutants containing deletions in their late leader region. All mutants produced more unspliced 19S RNA than did wild-type virus, and in two mutants, unspliced 19S RNA constituted more than 60% of the total 19S species. The other three mutants each utilized predominantly a different one of the three spliced species of 19S mRNA. All mutants also produced decreased quantities of 16S mRNA, indicating that they may be defective for splicing both late RNAs. None of the 5' termini of the 16S and 19S RNAs made by the five mutants predominated as in those made by the wild type. Some of the mutant 5' termini were the same as those used by the wild type, whereas others were different. Although present, the major 5'-end positions used by the wild type were frequently not used as major sites by the mutants. In addition, mutants with very similar deletion endpoints synthesized RNAs with different 5' ends. Thus, downstream mutations have a pronounced effect on the location of 5' ends of the late RNAs, and there is no obvious involvement of a measuring function in the placement of 5' ends. For all mutants and wild-type virus, the 5' termini used for 16S and 19S RNAs showed major differences, with some degree of correlation found between the 5' ends and the internal splices of specific mRNA species. A model for the regulation of simian virus 40 late gene expression is presented to explain these findings.     

Characterization and expression of novel singly spliced RNA species of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) expresses the Vif, Vpr, Vpu, and Env proteins through complex differential splicing of a single full-length RNA precursor. We used HIV-1-specific oligonucleotide primer pairs in a quantitative polymerase chain reaction procedure on RNA from fresh peripheral blood lymphocytes infected with HIV-1JR-CSF to detect and characterize the singly spliced RNA species which might encode these proteins. The nucleotide sequences at the junctions of splice donor and acceptor sites of these RNAs were determined. One of these RNAs, which has not been previously described, appears to be a novel HIV-1 RNA encoding Env and/or Vpu proteins.     

Intramolecular base pairing between the nematode spliced leader and its 5'' splice site is not essential for trans-splicing in vitro.

The spliced leader RNAs of both trypanosomes and nematodes can form similar secondary structures where the trans-splice donor site is involved in intramolecular base pairing with the spliced leader sequence. It has been proposed that this base pairing could serve to activate autonomously the SL RNA splice donor site. Here, we have examined exon requirements for trans-splicing in a nematode cell free system. Complete disruption of secondary structure interactions at and around the trans-splice donor site did not affect the ability of the SL RNA to function in trans-splicing. In addition, the highly conserved 22 nt sequence could be productively replaced by artificial exons ranging in size from 2 to 246 nucleotides. These results reinforce the view that the 'intron' portion of the SL RNA functions as an independent Sm snRNP whose role is to deliver exon sequences to the trans-spliceosome.     

Characterization of Two Simian Virus 40-Specific RNA Molecules from Infected BS-C-1 Cells

Two discrete simian virus 40 (SV40) RNA species sedimenting at 19 and 16S, respectively, that are present in infected BS-C-1 cells were characterized with respect to the base composition and the ribonuclease T1 fingerprints. The base composition of the 19S SV40 RNA was found to be cytidylic acid (C), 23.0; adenylic acid (A), 28.3; guanylic acid (G), 23.9; and uridylic acid (U), 24.8; that of the 16S SV40 RNA was C, 19.3; A, 34.0; G, 22.0; and U, 24.7 mol%. Analysis of the ribonuclease T1 fingerprints indicated a difference in the base sequence of the 19 and 16S SV40 RNA. The presence of long sequences of adenylic acid residues (poly A) in these viral RNAs was confirmed.     

Independent expression of the transforming amino-terminal domain of SV40 large I antigen from an alternatively spliced third SV40 early mRNA.

We found that simian virus 40 (SV40), in addition to the SV40 early proteins large T antigen (large T) and small antigen (small t), codes for a third early protein with a molecular weight of 17 kDa. This protein (17kT) is expressed from an alternatively spliced third SV40 early mRNA, using a splice donor site at position 4425 and a splice acceptor site at position 3679 of the SV40 genome. The 17kT protein consists of 135 amino acids. Of these, 131 correspond to the amino-terminus of large T, while the four carboxy-terminal amino acids are unique and encoded by a different reading frame. 17kT mRNA, and the corresponding protein, were found in all SV40 transformed cells analyzed, as well as in SV40 infected cells. Transfection of a cDNA expression vector encoding the 17kT protein into rat F111 fibroblasts induced phenotypic transformation of these cells. The expression of the transforming amino-terminal domain of large T as an independent 17kT protein might provide a means for individually regulating the various functions associated with this domain.