Influenza A virus encodes two segments (7 and 8) that produce mRNAs that can be spliced. We have investigated if naturally occurring sequence polymorphisms in the influenza A virus family affects splicing of these viral mRNAs, as that could potentially alter the NS1/NS2- and/or M1/M2-protein ratios. We compared splicing efficiency of segment 7 and 8 mRNAs of A/Brevig Mission/1918/1 (H1N1) and A/Netherlands/178/95 (H3N2), as well as various H5N1 avian strains. Results revealed that both segment 7 and 8 mRNAs of A/Brevig Mission/1918/1 (H1N1) were inefficiently spliced compared to other influenza virus segment 7 and 8 mRNAs. This resulted in production of higher levels of functional NS1 protein, which could potentially contribute to the pathogenic properties of the A/Brevig Mission/1918/1 (H1N1). We also show that A/Brevig Mission/1918/1 (H1N1) segment 8 mRNAs responded differently to overexpression of SR proteins than A/Netherlands/178/95 (H3N2).
Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/metabolism , RNA Splicing , Viral Nonstructural Proteins/biosynthesis , Viral Nonstructural Proteins/genetics , Amino Acid Sequence , Genes, Viral , HEK293 Cells , Humans , Influenza A Virus, H3N2 Subtype/genetics , Influenza A Virus, H3N2 Subtype/metabolism , Influenza A Virus, H5N1 Subtype/genetics , Influenza A Virus, H5N1 Subtype/metabolism , Plasmids , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , Viral Nonstructural Proteins/metabolism
We have previously shown that SRp55 inhibits splicing from HIV-1 exon 3, thereby generating partially spliced mRNAs encoding HIV-1 vpr. Here we show that SRp55 also inhibits splicing from HIV-1 exon 5 to generate HIV-1 vpu/env mRNA, albeit with lower efficiency. We also show that inhibition of HIV-1 splicing by SRp55 causes the appearance of partially spliced vpu, env and vpr mRNAs in the cytoplasm. SRp55 could also induce production of extracellular p24gag from a rev-defective HIV-1 provirus. These results indicate that SRp55 aids in the generation of partially spliced and unspliced HIV-1 mRNAs.
HIV-1/genetics , Nuclear Proteins/genetics , Phosphoproteins/genetics , RNA Splicing , Cells, Cultured , Cytoplasm/metabolism , Cytoplasm/virology , Exons , Genome, Viral , Human Immunodeficiency Virus Proteins/genetics , Human Immunodeficiency Virus Proteins/metabolism , Humans , Plasmids , Proviruses/genetics , RNA, Messenger , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Binding Proteins , Reverse Transcriptase Polymerase Chain Reaction , Serine-Arginine Splicing Factors , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/metabolism , env Gene Products, Human Immunodeficiency Virus/genetics , env Gene Products, Human Immunodeficiency Virus/metabolism , rev Gene Products, Human Immunodeficiency Virus/genetics , rev Gene Products, Human Immunodeficiency Virus/metabolism
HIV-1 non-coding exon 3 can either be spliced to exons 4, 4a, 4b, 4c, and 5 to generate tat, rev, and nef mRNAs or remain unspliced to produce the 13a7 vpr mRNA. Here we show that serine- and arginine-rich proteins 55 and 75 (SRp55 and SRp75) inhibit splicing from the 5'-splice site of exon 3 thereby causing an accumulation of the partially unspliced 13a7 vpr mRNA. In contrast, serine- and arginine-rich protein 40 (SRp40) induces splicing from exon 3 to exon 4, thereby promoting the production of the 1347 tat mRNA. We demonstrate that SRp55 stimulates vpr mRNA production by interacting with the previously identified HIV-1 splicing enhancer named GAR and inhibiting its function. This inhibition requires both serine arginine-rich and RNA-binding domains of SRp55, indicating that production of HIV-1 vpr mRNA depends on the interaction of SRp55 with an unknown factor.
Alternative Splicing/physiology , Exons/physiology , HIV-1/physiology , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , RNA Splice Sites/physiology , RNA, Messenger/metabolism , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , vpr Gene Products, Human Immunodeficiency Virus/biosynthesis , HeLa Cells , Humans , Nuclear Proteins/genetics , Phosphoproteins/genetics , RNA, Messenger/genetics , RNA, Viral/genetics , RNA-Binding Proteins/genetics , Serine-Arginine Splicing Factors , tat Gene Products, Human Immunodeficiency Virus/biosynthesis , tat Gene Products, Human Immunodeficiency Virus/genetics , vpr Gene Products, Human Immunodeficiency Virus/genetics
