Detailed analysis of the portion of the herpes simplex virus type 1 genome encoding glycoprotein C.

We previously showed that the right third of HindIII fragment L (0.59 to 0.65) of herpes simplex virus type 1 (HSV-1) encodes a family of mRNAs some members of which appear to be related by splicing. In the experiments described in this communication, we determined the nucleotide sequence of the DNA encoding this mRNA family and precisely located the mRNAs associated with this DNA sequence. The major mRNA species is unspliced and encoded by a 2.520-nucleotide region. Just upstream of the 5' end are TATA and CAT box sequences characteristic of HSV-1 promoters. The 3' end maps near a region containing a nominal polyadenylation signal. Three minor species (2,400, 2,200, and 1,900 bases, respectively) appear to share a very short leader sequence with the 5' end of the major mRNA and are then encoded by uninterrupted DNA sequences beginning about 100, 400, and 625 bases downstream of the 5' end of the major unspliced mRNA. These positions map at or very near positions which agree reasonably well with consensus splice acceptor sequences. The fourth mRNA is encoded by a contiguous 730-nucleotide sequence at the 3' end of the major unspliced mRNA and has its 5' end just downstream of recognizable TATA and CAT box sequences. We suggest that this mRNA is controlled by its own promoter. The nucleotide sequence data, in combination with the mRNA localization, demonstrate four potential polypeptides encoded by the region. The largest is 1,569 bases long and defines a 523-amino acid protein with sequence features characteristic of a glycoprotein. This was confirmed to be HSV-1 glycoprotein C by immune precipitation of the in vitro translation product of the major unspliced mRNA, performed with a polyspecific antibody to HSV-1 envelope glycoproteins (anti-env-1 serum), and by comparison of tryptic peptides of this translation product with those of authentic HSV-1 glycoprotein C. Polypeptides encoded by some of the minor species also were tentatively identified.     

Specific inactivation of inhibitory sequences in the 5' end of the human papillomavirus type 16 L1 open reading frame results in production of high levels of L1 protein in human epithelial cells

The expression of human papillomavirus type 16 late genes encoding virus capsid proteins L1 and L2 is restricted to terminally differentiated epithelial cells in the superficial layers of the squamous epithelium. We wish to understand the molecular mechanisms that determine the levels of expression of the human papillomavirus type 16 late genes. We have previously shown that the L1 coding region contains inhibitory sequences. Here we extend previous findings to show that the 5' end of the L1 gene contains strong inhibitory sequences but that the 3' end does not. We show that the first 514 nucleotides of the L1 coding region contain multiple inhibitory elements that act independently of one another and that the major inhibitory element is located within the first 129 nucleotides of the L1 gene. Introduction of point mutations in the inhibitory elements in the 5' end of the L1 gene which altered the RNA sequence without affecting the protein sequence specifically inactivated the inhibitory elements and resulted in production of high levels of human papillomavirus type 16 L1 mRNA and protein in human epithelial cells. Furthermore, we show that inhibitory sequences are present in the L1 coding regions of multiple human papillomavirus types, demonstrating that these elements are conserved among the human papillomaviruses, and suggest that they have an important function in the viral life cycle.     

Characterization of Herpes Simplex Virus Type 1 RNA Present in the Absence of De Novo Protein Synthesis

We used herpes simplex virus type 1 (HSV-1) DNA and restriction fragments of HSV-1 DNA covalently coupled to cellulose as a reagent to isolate for further characterization the major and minor HSV-1 immediate-early mRNA species in HeLa cells infected and maintained in the absence of de novo protein synthesis. Five major and several minor immediate-early mRNA species were characterized. One major species was a 4.2-kilobase mRNA mapping in the TR(S)/IR(S) region with its 3' end distal to the U(S) region; this mRNA encoded a 170,000-dalton polypeptide in vitro. A 2.8-kilobase mRNA, encoding a 120,000-dalton polypeptide, was mapped in the TR(L)/IR(L) region with its 3' end directed toward the U(L) region. Three 1.8-kilobase mRNA species were mapped. One, mapping in the IR(S) region with its 3' end in the U(S), encoded a 68,000-dalton polypeptide. One mapped in the TR(S) region and had its 3' end in the U(S) region; the third one encoded a 64,000-dalton polypeptide and mapped in the U(L) region near the IR(L) region. One minor species 5.2 kilobases in size was clearly detectable mapping in the U(L) region. Furthermore, there were indications that one or more immediate-early mRNA species approximately 3 kilobases in size hybridized to regions near the TR(L) and in or near the TR(S)/IR(S) regions. Nuclear immediate-early RNA mapped only in those regions where polyribosomal immediate-early mRNA mapped, although minor differences were seen. Finally, we demonstrated that at least three major immediate-early mRNA's-4.2 kilobases, 2.8 kilobases, and the 1.8-kilobase one mapping in the IR(S)/U(S) region-continued to appear on polyribosomes as functional mRNA late after infection.     

Identification of cis-acting intron and exon regions in influenza virus NS1 mRNA that inhibit splicing and cause the formation of aberrantly sedimenting presplicing complexes.

In in vitro splicing reactions, influenza virus NS1 mRNA was not detectably spliced, but nonetheless very efficiently formed ATP-dependent 55S complexes containing the U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins (snRNPs) (C. H. Agris, M. E. Nemeroff, and R. M. Krug, Mol. Cell. Biol. 9:259-267, 1989). We demonstrate that the block in splicing was caused by two regions in NS1 mRNA: (i) a large intron region (not including the branchpoint sequence) and (ii) an 85-nucleotide 3' exon region near the 3' end of the exon. After removal of both of these regions, the 5' and 3' splice sites and branchpoint of NS1 mRNA functioned efficiently in splicing, indicating that they were not defective. The two inhibitory regions shared one property: splicing inhibition was independent of the identity of the nucleotide sequence in either region. In other respects, however, the two inhibitory regions differed. The inhibitory activity of the intron region was proportional to its length, indicating that the inhibition was probably due to size only. In contrast, the 3' exon, which was of small size, was a context element; i.e., it functioned only when it was located at a specific position in the 3' exon of NS1 mRNA. To determine how these intron and exon regions inhibited splicing, we compared the types of splicing complexes formed by intact NS1 mRNA with those formed by spliceable NS1 mRNA lacking the intron and exon regions. Splicing complexes were formed by using purified splicing factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutational inactivation of two distinct negative RNA elements in the human papillomavirus type 16 L2 coding region induces production of high levels of L2 in human cells

Here we show that the 5' end and the middle region of the L2 coding sequence of human papillomavirus type 16 contain strong inhibitory RNA sequences termed inhibitory regions I and II. This is in contrast to L1, which contains one inhibitory region in the 5' end of the coding region. Inhibitory regions I and II acted in cis to reduce L2 mRNA levels and to inhibit the use of the mRNA. In tandem, the two regions reduced L2 mRNA production to undetectable levels. Specific mutational inactivation of the two inhibitory elements in the 5' end and in the middle region of L2 by the introduction of nucleotide substitutions that changed the nucleotide sequence but not the protein sequence resulted in production of high levels of L2 mRNA and protein. In contrast to L2, a partial L1 mutant in which only the first one third of L1 was mutated produced levels of L1 mRNA and protein similar to those in a full L1 mutant. In addition, the constitutive transport element of simian retrovirus type 1 overcomes the effect of the inhibitory sequences of L1 but not L2.     

Conditional mutants of the yeast mRNA capping enzyme show that the cap enhances, but is not required for, mRNA splicing.

The 5' end of eukaryotic mRNAs are modified by the addition of a 7-methyl guanosine (m7G) cap. The role of the cap in translation has been well established. Additionally, studies conducted in vitro or in microinjected Xenopus oocytes have implicated the cap in RNA processing and transport. To determine the fate of uncapped mRNA in intact yeast cells, conditional alleles of the gene encoding the capping enzyme guanylyltransferase subunit (CEG1) were generated. RNA analysis of temperature-sensitive ceg1 strains revealed an accumulation of unspliced pre-mRNAs and a corresponding decrease in spliced mRNAs at the restrictive temperature. A substantial proportion of spliced mRNA was also uncapped. Therefore, the cap appears to stimulate, but is not absolutely required for, splicing in vivo. In addition, steady-state levels of several mRNAs were decreased, perhaps due to increased degradation of uncapped mRNAs. In contrast to splicing, mRNA polyadenylation and transport to the cytoplasm were unaffected.     

Herpes simplex virus type 1 HindIII fragment L encodes spliced and complementary mRNA species.

We have used DNA bound to cellulose to isolate and translate in vitro herpes simplex virus type 1 (HSV-1) mRNA's encoded by HindIII fragment L (mapping between 0.592 and 0.647), and 8.450-base-pair (8.45-kb) portion of the long unique region of the viral genome. Readily detectable, late mRNA's 2.7 and 1.9 kb in size encoding 69,000- and 58,000-dalton polypeptides, respectively, were isolated. A very minor late mRNA family composed of two colinear forms, one 2.6 kb and one 2.8 kb, was isolated and found to encode only an 85,000-dalton polypeptide. A major early mRNA, 1.8 kb in size encoding a 64,000-dalton polypeptide, was also isolated. High-resolution mapping of these mRNA's by using S1 nuclease and exonuclease VII digestion of hybrids between them and 5' and 3' end-labeled DNA fragments from the region indicated that the major early mRNA contained no detectable splices, and about half of its 3' end was complementary to the 3' region of the very minor 2.6- to 2.8-kb mRNA's encoded on the opposite strand. These mRNA's also contained no detectable splices. The major late 2.7-kb mRNA was found to be a family made up of members with no detectable splices and members with variable-length (100 to 300 bases) segments spliced out very near (ca. 50 to 100 bases) the 5' end.     

Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo.

The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33 degrees C or lower than at 37 to 41 degrees C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41 degrees C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. We exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39 degrees C. However, after a short (about 30-min) lag following a shift to 33 degrees C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA. Increased splicing at this site in 54-5A4 viral RNA is probably driven by the unavailability of the usual 3' splice site for exon ligation. The thermosensitivity of this alternate splice event suggests that the sequences governing the thermodependence of MuSVts110 RNA splicing do not involve any particular 3' splice site or branch point sequence, but rather lie near the 5' end of the intron.     

Expression of the cytochrome b-URF6-URF5 region of the mouse mitochondrial genome

The nature of RNA coded by the only light-strand (L-strand) open-reading frame unidentified reading frame 6 (URF6) was studied by using a variety of single- and double-strand DNA subclones derived from the 3.6-kilobase (kb) cytochrome b (cyt b)-URF5 coding region of the mouse mitochondrial genome. Northern blot experiments using single-strand-specific M13 clones indicate that both the heavy (H) and L strands of this genomic region are symmetrically transcribed and processed into poly(adenylic acid) [poly(A)] RNAs of comparable size. The 1.2- and 2.4-kb RNAs coded by the H strand, putative mRNAs for cyt b and URF5 reading frames, respectively, are derived from a common precursor of 3.6-kb RNA. The L-strand-coded 1.15-kb RNA, on the other hand, is derived from a short-lived precursor of 3.6-kb RNA by a multiple-step processing involving a 2.4-kb intermediate RNA. The S1 nuclease protection experiments using both the 3'- or 5'-end-labeled DNA probes and also affinity-purified 32P-labeled RNA probes indicate that the 1.15-kb RNA maps between the start of the URF6 reading frame (3' end) and a region 590-600 nucleotides to the 5' end of this reading frame. The 1.15-kb RNA thus contains the entire URF6 coding sequence and an about 590-nucleotide-long 3' untranslated region. The molar abundance of the three mRNAs in the steady-state mitochondrial RNA varies markedly. The 1.15-kb URF6 mRNA is only one-tenth the level of 1.2-kb cyt b mRNA, although it is nearly as abundant as the 2.4-kb URF5 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)