The genes of influenza virus

The 5′ terminal sequences of several adenovirus 2 (Ad2) mRNAs, isolated late in infection, are complementary to sequences within the Ad2 genome which are remote from the DNA from which the main coding sequence of each mRNA is transcribed. This has been observed by forming RNA displacement loops (R loops) between Ad2 DNA and unfractionated polysomal RNA from infected cells. The 5′ terminal sequences of mRNAs in R loops, variously located between positions 36 and 92, form complex secondary hybrids with single-stranded DNA from restriction endonuclease fragments containing sequences to the left of position 36 on the Ad2 genome. The structures visualized in the electron microscope show that short sequences coded at map positions 16.6, 19.6 and 26.6 on the R strand are joined to form a leader sequence of 150–200 nucleotides at the 5′ end of many late mRNAs. A late mRNA which maps to the left of position 16.6 shows a different pattern of second site hybridization. It contains sequences from 4.9?6.0 linked directly to those from 9.6?10.9. These findings imply a new mechanism for the biosynthesis of Ad2 mRNA in mammalian cells.     

Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant.

The EIa region of an Adenovirus 5 recombinant has been substituted by a modular gene encoding dihydrofolate reductase (DHFR). In this recombinant, the mouse DHFR cDNA was positioned behind sequences of the major late promoter and the complete tripartite leader. The leader sequences end in the normal 5' splice site (SS) of the third leader, so that RNA splicing joins the tripartite leader to a 3' splice site immediately upstream of the DHFR cDNA. At late stages of infection, high levels of DHFR mRNAs were synthesized. At early times in the late stage, this mRNA was efficiently translated; however, at later times translation of DHFR decreased probably due to poor competition with other late mRNAs. Synthesis of DHFR protein from an analogous Adenovirus 5 recombinant containing only the first late leader was studied in parallel. Equivalent levels of DHFR mRNA were expressed after infection with this recombinant virus; however, the efficiency of DHFR translation was at least 20 fold lower than that of the DHFR mRNA containing the tripartite leader. This suggests that the tripartite leader sequence is important for translation in the late stage of infection. As reported previously, the Ad5 recombinant containing only the first leader vastly overexpresses polypeptide IX from a novel mRNA, formed by the splicing of the first leader in the modular DHFR gene to the 3' splice site in the EIb region. Cells infected with this recombinant synthesize very little normal mRNA from the EIb region. Here, we demonstrated that coinfection of 293 cells with this recombinant and wild type Adenovirus 5 also results in decreased EIb mRNA synthesis. We propose that the overproduction of polypeptide IX suppresses mRNA expression from the EIb and IX promoter sites, probably by an autoregulation loop active during lytic growth.     

Further mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments

RNA isolated from the cytoplasm of human cells at late times after infection by adenovirus type 2 (Ad2) has been fractionated by hybridization to fragments of Ad2 DNA which were produced by digestion with the restriction endonucleases Hpa I, Eco RI, Bam HI and Hind III. Cell-free translation of these partially purified mRNAs indicates that the genes for the late Ad2 proteins lie within the following intervals on the conventional Ad2 map: 15K (4.4–17.0 map units), IX and IVa₂ (7.5–17.0), IIIa (29.1–40.9), III and V (29.1–57.0), pVIII (40.9–57.0), pVI and II (40.9–70.7), 100K (59.0–83.4), pVIII (70.7–83.4) and IV (85.0–100). In addition to the primary hybridization of the late Ad2 mRNAs to the regions indicated above, most late Ad2 mRNAs (except those for 15K, IX and IVa₂) exhibited some hybridization to a secondary site between 17.0 and 29.1 map units.     

Secondary structure analysis of adenovirus tripartite leader

RNA secondary structure analysis was performed to understand the translation function of the adenovirus tripartite leader, a 200-nucleotide 5' noncoding region found on all late viral mRNAs. The tripartite leader facilitates the translation of viral mRNAs at late but not early times after infection and eliminates the normal requirement for the eukaryotic initiation factor 4F or cap binding protein complex. Secondary structures were determined by probing 5' or 3' end-labeled tripartite leader RNAs under nondenaturing conditions with various single strand-specific nucleases, and the information was used to generate a potential model structure. The resulting structure is attractive since it may explain the unusual translation behavior conferred by the tripartite leader. We demonstrate that the first leader segment is predominantly single-stranded, a property consistent with the ability to enhance translation and provide independence from cap binding protein complex. In contrast, the remaining two leader segments form a moderately stable base-paired structure, except for a large hairpin loop. To confirm these findings, the secondary structure of the tripartite leader was also probed when it was attached to a large segment of a messenger RNA and was found to be very similar to that of the individual leader RNA. These findings suggest several possible mechanisms to account for the translation activity of the tripartite leader.     

Translation by the adenovirus tripartite leader: elements which determine independence from cap-binding protein complex.

The adenovirus tripartite leader is a 200-nucleotide-long 5' noncoding region which facilitates translation of viral mRNAs at late times after infection. The tripartite leader also confers the ability to initiate translation independent of the requirement for cap-binding protein complex or eIF-4F without any requirement for adenovirus gene products. To elucidate the manner by which the tripartite leader functions, the primary determinants of leader activity were investigated in vivo by testing a series of mutations expressed from transfected plasmids. The results of these experiments indicate that the tripartite leader does not promote internal ribosome binding, at least in a manner recently described for picornavirus mRNAs. In addition, despite an unusual arrangement of sequences complementary to the 3' end of 18S rRNA in the tripartite leader, we could find no evidence for involvement in its translation activity. Instead, our results are consistent with a model in which much of the first leader is maintained in an unstructured conformation which determines the ability of the tripartite leader to facilitate translation and bypass a normal requirement for eIF-4F activity. Several possible translation models are discussed, as well as the implications for translation of late viral mRNAs.