Lance, there is not a striking difference between the two secondary
Lance, there is not a striking difference between the two secondary structures generated, despite the 7 purchase 3-MA nucleotides that differ between the A8 and 57 sequences. The most visible changes actually occur upstream from the polyadenylation signal, where the 1st, 2nd, and 3rd nucleotides are incorporated into a stem structure in the A8 sequence, thereby lengthening the stem structure compared to the 57 sequence. The site with the smallest conformational change contains the 5th, 6th and 7th nucleotides. These three nucleotides reside within a stem-loop structure that protrudes out into the PBS.Choo et al. Virology Journal 2013, 10:124 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27196668 http://www.virologyj.com/content/10/1/Page 7 ofFigure 5 Luciferase activity of mutation series vectors. A series of vectors where the sequences from A8 were gradually mutated into 57 sequences were constructed and their luciferase activities were quantified. Mutations from A8 to 57 are indicated by triangles. The mean values from 4? independent experiments and the SEM are shown. Statistical comparison was done using the t test. ns: differences were not significant. *: differences were not significant versus R7f-L.The possible roles played by these nucleotides are discussed further in the next section.Alignment of the 0.3-kb fragment sequences among gamma retrovirusesSince point mutational analysis indicated the 1st to 7th nucleotides contribute to the luciferase activities of the vectors, we compared the sequences including these nucleotides in gamma retroviruses containing MLVs, Feline leukemia virus (FLV), and Gibbon ape leukemia virus (GALV) (Table 1). The PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28607003 1st guanine (G) nucleotide in A8 was well conserved among these gamma retroviruses except for 57. The 2nd and 3rd nucleotides in A8 were adenine (A) and thymidine (T), respectively, and the 4th, 5th, and 6th nucleotides in 57 were G, cytosine (C), and T, respectively. These nucleotides were relatively conserved among the gamma retroviruses that were analyzed. The 7th guanine nucleotide in A8 was well conserved in not only MLVs but also in FLV and the GALV. Among the sequences analyzed, only the Fr-MLV clone 57 virus had an adenine at the 7th nucleotide.Discussion In the present study, to investigate the role of a 0.3-kb KpnI-AatII fragment containing the R-U5-5’leader sequence, recombinant luciferase vectors were constructed by replacing the viral-env-gene with the luc-gene in proviral sequences to produce R7f-L and Rec5-L (Figure 2A). As shown in Figure 2B, R7f-L exhibited about 2 times higher luciferase expression compared to Rec5-L. This result agrees well with experiments that utilized the chimeric viruses R7f and Rec5, in which the Env protein expression level of R7f-infected cells was higher than that of Rec5-infected cells [5]. Therefore, the experimental system using R7f-L and Rec5-L vectors is useful to analyze the function of the 0.3-kb fragment in Envprotein expression. Next, to examine whether the 0.3-kb fragment functions in the 5’LTR-leader sequence and/or in the 3’LTR, we constructed R7fa-L and R7fb-L. R7fa-L contains the 0.3-kb fragment of A8 sequences only at the 5’LTR, and R7fb-L contains the 57 sequences at the 5’LTR but has the A8 sequences of the R-U5 region at the 3’LTR (Figure 2A). The results of a luciferase assay showed that R7fa-L mimics the expression level of R7f-L (Figure 2B). R7fb-L, despite having partial A8 sequences at its 3’LTR, had a similarly reduced expression level of Rec5-L. These results suggested that luciferase expre.