Regulation of the extent of splicing of influenza virus NS1 mRNA: role of the rates of splicing and of the nucleocytoplasmic transport of NS1 mRNA.

Influenza virus NS1 mRNA is spliced by host nuclear enzymes to form NS2 mRNA, and this splicing is regulated in infected cells such that the steady-state amount of spliced NS2 mRNA is only about 10% of that of unspliced NS1 mRNA. This regulation would be expected to result from a suppression in the rate of splicing coupled with the efficient transport of unspliced NS1 mRNA from the nucleus. To determine whether the rate of splicing of NS1 mRNA was controlled by trans factors in influenza virus-infected cells, the NS1 gene was inserted into an adenovirus vector. The rates of splicing of NS1 mRNA in cells infected with this vector and in influenza virus-infected cells were measured by pulse-labeling with [3H]uridine. The rates of splicing of NS1 mRNA in the two systems were not significantly different, strongly suggesting that the rate of splicing of NS1 mRNA in influenza virus-infected cells is controlled solely by cis-acting sequences in NS1 mRNA itself. In contrast to the rate of splicing, the extent of splicing of NS1 mRNA in the cells infected by the adenovirus recombinant was dramatically increased relative to that occurring in influenza virus-infected cells. This could be attributed largely, if not totally, to a block in the nucleocytoplasmic transport of unspliced NS1 mRNA in the recombinant-infected cells. Most of the unspliced NS1 mRNA was in the nuclear fraction, and no detectable NS1 protein was synthesized. When the 3' splice site of NS1 mRNA was inactivated by mutation, NS1 mRNA was transported and translated, indicating that the transport block occurred because NS1 rRNA was committed to the splicing pathway. This transport block is apparently obviated in influenza virus-infected cells. These experiments demonstrate the important role of the nucleocytoplasmic transport of unspliced NS1 mRNA in regulating the extent of splicing of NS1 mRNA.     

Influenza B virus genome: sequences and structural organization of RNA segment 8 and the mRNAs coding for the NS1 and NS2 proteins.

Double-stranded DNA derived from influenza B virus genome RNA segment 8, which codes for the NS1 and NS2 proteins, was constructed by hybridization of full-length cDNA copies of RNA segment 8 and of the NS1 mRNA. This DNA was cloned in plasmid pBR322 and sequenced. The NS1 mRNA (approximately 1,080 viral nucleotides) contains nonviral nucleotides at its 5' end and is capable of coding for a protein of 281 amino acids. Sequencing of the NS2 mRNA has shown that it contains an interrupted sequence of 655 nucleotides and is most likely synthesized by a splicing mechanism. The first approximately 75 virus-specific nucleotides at the 5' end of the NS2 mRNA are the same as are found at the 5' -end of the NS1 mRNA. This region contains the initiation codon for protein synthesis and coding information for 10 amino acids common to the two proteins. The approximately 350-nucleotide body region of the NS2 mRNA can be translated in the +1 reading frame, and the sequence indicates that the NS1 and NS2 protein-coding regions overlap by 52 amino acids translated from different reading frames. Thus, between the influenza A and B viruses, the organization of the NS1 and NS2 mRNAs and the sizes of the NS2 mRNA and protein are conserved despite the larger size of the influenza B virus RNA segment, NS1 mRNA, and NS1 protein.     

Cellular RNA Binding Proteins NS1-BP and hnRNP K Regulate Influenza A Virus RNA Splicing

Influenza A virus is a major human pathogen with a genome comprised of eight single-strand, negative-sense, RNA segments. Two viral RNA segments, NS1 and M, undergo alternative splicing and yield several proteins including NS1, NS2, M1 and M2 proteins. However, the mechanisms or players involved in splicing of these viral RNA segments have not been fully studied. Here, by investigating the interacting partners and function of the cellular protein NS1-binding protein (NS1-BP), we revealed novel players in the splicing of the M1 segment. Using a proteomics approach, we identified a complex of RNA binding proteins containing NS1-BP and heterogeneous nuclear ribonucleoproteins (hnRNPs), among which are hnRNPs involved in host pre-mRNA splicing. We found that low levels of NS1-BP specifically impaired proper alternative splicing of the viral M1 mRNA segment to yield the M2 mRNA without affecting splicing of mRNA₃, M4, or the NS mRNA segments. Further biochemical analysis by formaldehyde and UV cross-linking demonstrated that NS1-BP did not interact directly with viral M1 mRNA but its interacting partners, hnRNPs A1, K, L, and M, directly bound M1 mRNA. Among these hnRNPs, we identified hnRNP K as a major mediator of M1 mRNA splicing. The M1 mRNA segment generates the matrix protein M1 and the M2 ion channel, which are essential proteins involved in viral trafficking, release into the cytoplasm, and budding. Thus, reduction of NS1-BP and/or hnRNP K levels altered M2/M1 mRNA and protein ratios, decreasing M2 levels and inhibiting virus replication. Thus, NS1-BP-hnRNPK complex is a key mediator of influenza A virus gene expression.     

Inhibition of c-erbA mRNA splicing by a naturally occurring antisense RNA.

The rat erbA alpha locus encodes two overlapping mRNAs, alpha 1 and alpha 2, which are identical except for their most 3' exons. alpha 1 mRNA encodes a thyroid hormone receptor, while alpha 2 encodes an altered ligand binding domain of unknown function. Previous studies have shown that the ratio of alpha 1 to alpha 2 is highest in cells expressing a high level of a third RNA, Rev-ErbA alpha mRNA, which is transcribed in the opposite direction and is complementary to alpha 2 but not alpha 1 mRNA. It was hypothesized that base pairing with Rev-ErbA alpha blocks splicing of alpha 2 mRNA, thereby favoring formation of the non-overlapping alpha 1. To test this model, a system was developed in which alpha 2 pre-mRNAs were accurately spliced in vitro. Splicing was inhibited by the addition of a 5-fold excess of antisense RNAs containing the 3' end of Rev-ErbA alpha mRNA. Both an antisense RNA extending across the 3' splice site and a shorter RNA complementary only to exon sequences efficiently blocked splicing. However, splicing was only inhibited by complementary RNAs. These observations are consistent with a mechanism in which base pairing with a complementary RNA regulates alternative processing of alpha 1 and alpha 2 mRNAs.     

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.     

A primer vector system that allows temperature dependent gene amplification and expression in mammalian cells: regulation of the influenza virus NS1 gene expression.

Influenza virus RNA segment 8 has been cloned into primer-vector pSLts1. This vector was designed to replicate in simian cells in a temperature dependent fashion by use of the SV40 tsA209 T-antigen gene. The oriented synthesis of cDNA on dT-tailed pSLts1 was performed on in vitro synthesized mRNA, and the second DNA strand was primed with an influenza-specific terminal oligodeoxynucleotide. Recombinant pSLVa232 contained the RNA segment 8 sequence directly fused to the SV40 late promoter contained in pSLts1, and followed by the SV40 polyadenylation signal. Expression of NS1 gene in transfected COS cells took place at a level comparable to that found in infected cells. When VERO cell cultures were transfected with recombinant pSLVa232, expression of the NS1 gene was temperature dependent. Close to one hundred fold increase in the amplification and expression of the cloned gene was observed after shift down of the transfected cells to permissive temperature. Vector pSLts1 and the cloning strategy described may be useful for the specific cloning and regulated expression of mRNAs of known 5'-terminal sequence.     

Evidence that the matrix protein of influenza C virus is coded for by a spliced mRNA.

In contrast to influenza A and B viruses, which encode their matrix (M) proteins via an unspliced mRNA, the influenza C virus M protein appears to be coded for by a spliced mRNA from RNA segment 6. Although an open reading frame in RNA segment 6 of influenza C/JJ/50 virus could potentially code for a protein of 374 amino acids, a splicing event results in an mRNA coding for a 242-amino-acid M protein. The message for this protein represents the major M gene-specific mRNA species in C virus-infected cells. Despite the difference in coding strategies, there are sequence homologies among the M proteins of influenza A, B, and C viruses which confirm the evolutionary relationship of the three influenza virus types.     

Oligonucleotide-targeted degradation of U1 and U2 snRNAs reveals differential interactions of simian virus 40 pre-mRNAs with snRNPs.

We have investigated the roles of U1 and U2 snRNP particles in SV40 pre-mRNA splicing by oligonucleotide-targeted degradation of U1 or U2 snRNAs in Xenopus laevis oocytes. Microinjection of oligonucleotides complementary to regions of U1 or U2 RNAs either in the presence or absence of SV40 DNA resulted in specific cleavage of the corresponding snRNA. Unexpectedly, degradation of U1 or U2 snRNA was far more extensive when the oligonucleotide was injected without, or prior to, introduction of viral DNA. In either co-injected or pre-injected oocytes, these oligonucleotides caused a dramatic reduction in the accumulation of spliced SV40 mRNA expressed from the viral late region, and a commensurate increase in unspliced late RNA. When pre-injected, two different U2 specific oligonucleotides also inhibited the formation of both large and small tumor antigen spliced early mRNAs. However, even when, by pre-injection of a U1 5' end-specific oligonucleotide, greater than 95% degradation of the U1 snRNA 5' ends occurred in oocytes, no reduction in early pre-mRNA splicing was observed. In contrast, the same U1 5' end oligonucleotide, when added to HeLa splicing extracts, substantially inhibited the splicing of SV40 early pre-mRNA, indicating that U1 mRNP is not totally dispensable for early splicing. These findings confirm and extend our earlier observations which suggested that different pre-mRNAs vary in their requirements for snRNPs.     

Relationship between the messenger RNAs transcribed from two overlapping genes of influenza virus.

The relationship of the mRNAs encoding the NS1 and NS2 polypeptides of influenza virus has been investigated through synthesis and characterisation of complementary DNA copies of the mRNAs. Previous work had shown that both mRNAs are encoded by virion RNA segment 8, and that the sequences comprising the smaller of the two mRNAs (the NS2 mRNA) were also present on the NS1 mRNA. Our results indicate that the mRNA encoding the NS2 polypeptide of the avian influenza, fowl plague virus, is approximately 400 ntds long, and that its sequences correspond largely with the 3'-terminal region of the NS1 mRNA.