The effect of specific mutations at and around thegag-polgene junction of Moloney murine leukaemia virus

Abstract

By carrying out oligonucleotide-directed mutagenesis, in vitro, on a 3.3 kb XhoI-HindIII fragment from Moloney murine leukaemia virus Mo-MuLV proviral DNA, inserted into the phagemid pTZ19R, nine separate fragments have been prepared in which mutations have been inserted at and around the gag-pol gene junction. Using these mutant fragments Mo-MuLV proviral DNA has been reassembled and cloned into pBR322. Examination of the mutant proviral DNAs in mouse culture cells indicates that a terminator codon at the gag-pol junction is essential for function, but any of the three chain terminator codons gives an active virus. Also the region of secondary structure surrounding the terminator codon must be preserved.

Identification of amino acids inserted during suppression of UAA and UGA termination codons at the gag-pol junction of Moloney murine leukemia virus.

“…These results demonstrated that the suppression signals are not specific for UAG and that mammalian cells and cell extracts contain tRNAs capable of suppressing UGA and UAA codons which appear in a retroviral context. Similar findings have been reported by other investigators working with tobacco mosaic virus (TMV) (7), Sindbis virus (8), and MuLV (9).…”

Analysis of leaky viral translation termination codons in vivo by transient expression of improved ?-glucuronidase vectors

“…Alternatively, readthrough over these stops could involve mechanisms that require more extensive sequence elements than we have tested here. Recently, it has been shown that viral reproduction is inhibited by mutations which disrupt a potential stem and loop structure around the suppressible amber stop codon of Moloney murine leukaemia virus [28]. Although regions with substantial secondary structure can be identified around the leaky termination regions of CarMV, MCMV and TRV (not shown), the inclusion of these sequences in our constructs (pMCMV-Am and pTRV-Op) was not sufficient to direct detectable readthrough.…”

Evidence that a downstream pseudoknot is required for translational read-through of the Moloney murine leukemia virus gag stop codon.

“…This result still holds when gag is provided in trans (13). In contrast, substitution of the UAG terminator by either of the other stop codons, UAA or UGA, surprisingly gives a similar level of read-through to that seen with UAG (15) and results in productive infection (14). As expected, glutamine is not inserted in response to the UGA codon (16) and the utilization of tRNAs other than tRNAGIn hints that some feature of the RNA in the vicinity of the stop codon is a general stimulator of stop codon read-through.…”

“…The read-through product, the gag-pol fusion polyprotein with glutamine inserted at the stop codon (10,11), is the only source of reverse transcriptase (and other pol products), since the mRNA is organized so that ribosomes cannot directly enter at the start of the pol gene. The ratio of gag to gag-pol polyproteins may be critical, since substitution of the "leaky" UAG terminator with a CAG glutamine codon resulted in the inability to produce virus (13,14). This result still holds when gag is provided in trans (13).…”

“…la), and it was suggested that the "flanking" stem-loop of that structure (nt -21 to + 18) might be important for read-through (9,20). Evidence that the flanking stem is important for productive infection has been obtained by comparing single mutants to pairs of compensating mutants in the putative stem (14). However, these studies do not distinguish between an effect on stop codon read-through and involvement of the stem in some other viral function.…”