Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2000 Apr 1;28(7):1625-34.
doi: 10.1093/nar/28.7.1625.

Initiation of translation by non-AUG codons in human T-cell lymphotropic virus type I mRNA encoding both Rex and Tax regulatory proteins

S Corcelette  1 , T Massé, J J Madjar
Affiliations
Comparative Study

Initiation of translation by non-AUG codons in human T-cell lymphotropic virus type I mRNA encoding both Rex and Tax regulatory proteins

S Corcelette et al. Nucleic Acids Res. .

Abstract

Human T-cell lymphotropic virus type I (HTLV-I) double-spliced mRNA exhibits two GUG and two CUG codons upstream to, and in frame with, the sequences encoding Rex and Tax regulatory proteins, respectively. To verify whether these GUG and CUG codons could be used as additional initiation codons of translation, two chimeric constructs were built for directing the synthesis of either Rex-CAT or Tax-CAT fusion proteins. In both cases, the CAT reporter sequence was inserted after the Tax AUG codon and in frame with either the Rex or Tax AUG codon. Under transient expression of these constructs, other proteins of higher molecular mass were synthesized in addition to the expected Rex-CAT and Tax-CAT proteins. The potential non-AUG initiation codons were exchanged for either an AUG codon or a non-initiation codon. This allowed us to demonstrate that the two GUG codons in frame with the Rex coding sequence, and only the second CUG in frame with the Tax coding sequence, were used as additional initiation codons. In HTLV-I infected cells, two Rex and one Tax additional proteins were detected that exhibited molecular mass compatible with the use of the two GUG and the second CUG as additional initiation codons of translation. Comparison of the HTLV-I proviral DNA sequence with that of other HTLV-related retroviruses revealed a striking conservation of the three non-AUG initiation codons, strongly suggesting their use for the synthesis of additional Rex and Tax proteins.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Structure of HTLV-I double- and single-spliced mRNA. (A) HTLV-I double-spliced mRNA results from addition of exon I, the sequence of which is entirely in the 5′ UTR, then exon II bearing two GUG, two CUG and the Rex and Tax AUG initiation codons, and finally exon III containing, first, Rex then Tax stop codons. The Rex-responsive element (XRE) is a 256 nt long sequence located at the 3′ end of all HTLV-I mRNA species. The two GUG codons at +217 and +226 are in frame with the Rex coding sequence that begins at AUG250; the two CUG codons at +210 and +246 are in frame with the Tax coding sequence that begins at AUG306. (B) HTLV-I single-spliced mRNA. Translation initiated by AUG250 potentially directs the synthesis of a putative short peptide due to the presence of an in-frame stop codon (UGA328) in the Env coding sequence. Translation initiated by AUG306 directs the synthesis of the envelope glycoprotein, the coding sequence of which is entirely in intron II. Nucleotide triplets are numbered from the mRNA cap site and according to the position of their first nucleotide. The nucleotide sequence is identical in both the single- and double-spliced mRNA, up to the G immediately following AUG306. In both mRNA species (A and B) the part originating from exon I is 118 nt long.
Figure 2
Figure 2
Chimeric mRNA synthesized by transcription of pHTL-Rex–CAT and pHTL-Tax–CAT. The CAT coding sequence devoid of its initiation codon was inserted in a plasmid derived from pHLX-I (33), downstream from AUG306 and in frame with either Rex AUG initiation codon (AUG250) to give pHTL-Rex–CAT (A), or Tax AUG initiation codon (AUG306) to give pHTL-Tax–CAT (B). (A) Rex–CAT mRNA directs the synthesis of a 240 amino acid long Rex–CAT protein by translation initiated by AUG250. This chimeric Rex–CAT protein contains the first 19 amino acids of Rex protein. In this 19 amino acid long sequence are located both nuclear and nucleolar localization signals (NLS/NoLS) and the binding domain of Rex to the XRE. Due to the presence of a stop codon (UGA345) in frame with AUG306, translation initiated by AUG306 would direct the synthesis of a putative 13 amino acid long peptide. (B) Tax–CAT mRNA directs the synthesis of a 221 amino acid long Tax–CAT protein by translation initiated by AUG306. Due to the presence of a stop codon (UAA445) in frame with AUG250, translation initiated by AUG250 would direct the synthesis of a putative 65 amino acid long peptide containing the first 19 amino acids of Rex protein.
Figure 3
Figure 3
Detection of several CAT-fusion proteins in HeLa cells transiently expressing either pHTL-Rex–CAT or pHTL-Tax–CAT. After transfection of HeLa cells with either pHTL-Rex–CAT or pHTL-Tax–CAT, total proteins were separated by SDS–PAGE, then probed by western blot using an anti-CAT antibody. The apparent molecular mass of each Rex–CAT (left) and Tax–CAT (right) protein is indicated in kDa.
Figure 4
Figure 4
Identification of three initiation codons directing the synthesis of Rex–CAT proteins. HeLa cells were transfected with either wild-type or modified pHTL-Rex–CAT in which putative AUG and GUG initiation codons were submitted to site-directed mutagenesis. AUG250 was exchanged for AUU and the two GUG (217 and 226) for either GUA or AUG. In each case, total HeLa cell proteins were separated by SDS–PAGE, then probed by western blot for detecting Rex–CAT proteins. (A) AUG250 was exchanged for AUU (lane 2). (B) GUG217 was exchanged for either GUA (lane 2) or AUG (lane 3). (C) GUG226 was exchanged for either GUA (lane 2) or AUG (lane 3).
Figure 5
Figure 5
Identification of two initiation codons directing the synthesis of Tax–CAT proteins. HeLa cells were transfected with either wild-type or modified pHTL-Tax–CAT in which putative AUG and CUG initiation codons were submitted to site-directed mutagenesis. AUG306 was exchanged for UUG, CUG210 was exchanged for either CUU or AUG and CUG246 was exchanged for either CCG or AUG. In each case, total HeLa cell proteins were separated by SDS–PAGE, then probed by western blot for detecting Tax–CAT proteins. (A) AUG306 was exchanged for UUG (lane 2). (B) CUG210 was exchanged for either CUU (lane 2) or AUG (lane 3). Position of the Tax–CAT protein resulting from a translation initiated by AUG210 is indicated by an arrow to the right of lane 3. (C) CUG246 was exchanged for either CCG (lane 2) or AUG (lane 3).
Figure 6
Figure 6
Influence of AUG250 and CUG nucleotide context on translation initiated by CUG246. HeLa cells were transfected with either wild-type pHTL-Tax–CAT (lane 1) or the same construct in which AUG250 was exchanged for either AUU (lane 2) or UAG (lane 3). In each case, total HeLa cell proteins were separated by SDS–PAGE, then probed by western blot to detect Tax–CAT proteins.
Figure 7
Figure 7
Detection of different Rex proteins in C91PL and MT-2 cells. Total proteins from C91PL and MT-2 cells, as well as from Jurkat and HeLa cells used as controls, were separated by SDS–PAGE (12%) then transferred onto a nitrocellulose membrane. Immunodetection of HTLV-I proteins by western blot was performed using the serum of a patient infected with HTLV-I. Positions of the putative Rex proteins are specified by their molecular mass to the right of the figure. Positions of protein markers of known molecular mass are indicated in kDa to the left of the figure.
Figure 8
Figure 8
Detection of an additional Tax protein. Total proteins from C91PL and MT-2 cells as well as from pCMV-2 transfected HeLa cells were separated by SDS–PAGE (10%), then transferred onto a nitrocellulose membrane. Immunodetection of Tax proteins by western blot was performed using a polyclonal antibody directed against Tax protein (anti-Tax). Non-immune serum (NI) was used as control. (A) C91PL cells. (B) MT-2 cells. (C) HeLa cells harvested 24 and 36 h after transfection with pCMV-2, a construct directing the synthesis of an mRNA identical to the HTLV-I double-spliced mRNA species. The apparent molecular mass of Tax proteins that were specifically detected with the anti-Tax antibody are indicated to the right of the figure.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Nagashima K., Yoshida,M. and Seiki,M. (1986) J. Virol., 60, 394–399. - PMC - PubMed
    1. Cullen B.R. (1992) Microbiol. Rev., 56, 375–394. - PMC - PubMed
    1. Grassmann R., Berchtold,S., Aepinus,C., Ballaun,C., Boehnlein,E. and Fleckenstein,B. (1991) J. Virol., 65, 3721–3727. - PMC - PubMed
    1. Siomi H., Shida,H., Nam,S.H., Nosaka,T., Maki,M. and Hatanaka,M. (1988) Cell, 55, 197–209. - PubMed
    1. Cullen B.R. (1998) Virology, 249, 203–210. - PubMed

Publication types

MeSH terms