monash_128299.pdf (3.49 MB)
Download file

Post-transcriptional regulation of HIV-1 asp: Potential control by a series of short open reading frames

Download (3.49 MB)
posted on 23.02.2017, 01:00 by Barbagallo, Michael Salvatore
The positive sense strand of the HIV-1 genome encodes nine different proteins. These include structural proteins (Gag, Pol and Env), regulatory proteins (Tat and Rev) as well as accessory proteins (Vpu, Vpr, Vif and Nef). In addition to the nine positive sense genes, a negative sense gene, asp, has been identified opposite in orientation to env. Bioinformatic analyses suggest that asp encodes a hydrophobic, membrane associated protein of 189 aa. Negative sense transcription, regulated by LTR sequences, has been observed early in HIV-1 infection in vitro. However the mechanism of asp expression and function of the putative ASP protein still remain unclear. In some viral strains a series of six short open reading frames (sORFs) positioned upstream of the asp gene have the potential to regulate asp expression. This thesis examines the role of these sORFs in control of expression of downstream genes. All subtypes of HIV-1 were examined to detect the negative sense asp ORF, and to identify potential regulatory sequences. A series of strongly conserved upstream sORFs was identified. The sORF series was particularly well conserved amongst the A, B, C and D clade strains with sORFs I, V and VI being highly conserved across all the subtypes examined. This potential control region from HIV-1NL4-3, containing six sORFs, was cloned upstream of the reporter gene EGFP. Expression by transfection of HEK293 cells indicated that the introduction of this sORF region inhibits EGFP reporter expression; analysis of transcripts revealed no significant change in levels of EGFP mRNA, suggesting that regulation occurs post-transcription. RT-PCR analysis of transcripts further demonstrated that the upstream sORF region undergoes alternative splicing in vitro. The most abundant product (Spliced Variant 1) is spliced to remove sORFs I to V, leaving only the in-frame sORF VI. Sequence analysis revealed the presence and high conservation of typical splice donor and acceptor site motifs. Spliced Variant 2, containing sORFs I, II and VI; utilised a lesser well conserved donor in conjunction with the splice acceptor common to Spliced Variant 1. Cloning of Spliced Variant 2 enabled the detection of a third product, Spliced Variant 3. This Spliced Variant (3) presented an alternate initiation codon for sORF VI, designated VIalt. While Spliced Variants 1 and 2 inhibited, to varying extents, downstream expression; Spliced Variant 3 permitted expression. Mutation of the highly conserved splice donor and acceptor sites modulates, but does not fully relieve, inhibition of reporter EGFP production. These data were further supported by sequential mutation of the sORF initiation codons in which, to varying levels, each mutation alleviated the inhibitory nature of the sORF series, suggesting a translational mechanism for the control of asp expression. Toeprinting analysis of the sORF region also revealed the potential for ribosomes to initiate at sORFs I, II, IV, VI and VIalt, yet only weak toeprints were observed for sORF III and sORF V. Initiation at a cryptic CUG codon located 14 nucleotides downstream from sORF VIalt was also detected. These data suggest that the leaky scanning and/or termination reinitiation mechanisms of translation account for the mode of translation across the sORF transcript, and that sORF VI, alone, inhibits downstream translation. Upstream sORFs engage the ribosome, facilitating subsequent initiation downstream at sORF VI. Alternative splicing determines the presence or absence of upstream sORFs, therefore the efficiency of recognition of the sORF VI initiation codon and the degree of inhibition of downstream gene expression. These findings suggest a complex mechanism, involving both splicing and translational control, modulate asp gene expression. The strong conservation of asp and its sORFs across all HIV-1 subtypes suggests that the asp gene product may have a role in the pathogenesis of HIV-1 and requires tight regulation. This study promotes further examination of the negative sense transcript and its function in the HIV-1 viral life cycle.


Campus location


Principal supervisor

Jennifer Mosse

Year of Award


Department, School or Centre

School of Applied Sciences and Engineering (Gippsland)


Doctor of Philosophy

Degree Type



Faculty of Science