Posttranscriptional regulation of gene expression in retroviruses and human papillomaviruses

Abstract

Human immunodeficiency virus type I (HIV-1) is the prototype of complex retroviruses, whereas human papillomaviruses (HPVs) are nonenveloped, cricular double-stranded DNA viruses. The HIV-1 Rev protein is a posttranscriptional regulator, which acts on its target RNA sequence termed the Rev-responsive element (RRE) to facilitate nuclear export of unspliced and partially spliced HIV-1 mRNAs, and to improve their stability and translatability. Although HPVs are distinct from HIV-1, gene expression of HPVs may also be posttranscriptionally regulated since replication of HPV and HIV can be divided into an early regulatory phase and a late, structural phase. Interaction of Rev and RRE provides a useful test system to study posttranscriptional regulation of gene expression in complex retroviruses as well as in DNA viruses, such as HPVs.

Using the HIV-1 tat mRNAs, we demonstrate that the strength of the tat AUG and the length of the tat ORF are the major factors that substantially affect translation efficiency of the downstream rev ORF, whereas intercistronic distance influence reinitiation of the rev ORF to a lower extent. The presence of the tat ORF strongly inhibited translation of the downstream rev ORF. This may reflect the requirement for high levels of Tat at an early stage of the virus life cycle, and may avoid a premature switch from early to late phase of gene expression to prevent inefficient virus production. Unlike HIV-1, we did not find inhibitory RNA sequences in the gag coding region of equine infectious anemia virus (EIAV). However, sequences upstream of the gag translational start codon strongly inhibited Gag production in the absence of Rev and rendered Gag production Rev dependent. The inhibitory effect could be overcome by Rev and RRE or the constitutive transport element (CTE) of simian retrovirus type I (SRV-I). The minimal inhibitory sequence coincides with the EIAV major 5' splice site. Our results demonstrated that both integrity and location of the EIAV 5' splice site are required for strong inhibition of EIAV Gag production. Late gene expression of HPVs is tightly linked to the stage of differentiation of epithelial cells. We found that the HPV-16 L1 coding region contains intragenic RNA inhibitory sequences, which repressed L1 production at the posttranscriptional level. Production of the HPV-16 Ll protein could be activated by Rev and RRE or SRV-I CTE. A major inhibitory element was localized between nucleotides 5813 and 6150. The presence of inhibitory sequences on the HPV-16 late mRNAs may explain the lack of L1 production in nondifferentiated epithelial cells.

The HPV-1 late 3' untranslated region (UTR) also contains inhibitory sequences, which act posttranscriptionally to inhibit gene expression in an orientation-dependent manner. The inhibition is caused, at least in part, by reducing mRNA levels and by inefficient utilization of mRNAs. The inhibition was counteracted by HIV-1 Rev and RRE or SRV-I CTE, or could be bypassed by transcription in the cytoplasm using a vaccinia virus-T7 RNA polymerase-based expression system. The major inhibitory element was localized to an AU-rich sequence between nucleotides 6943 and 7014 near the 5' end of the HPV-1 late 3' UTR. We proposed that cellular factors may bind to the HPV-1 inhibitory RNA sequence, resulting in inhibition of HPV-1 late gene expression. Using RNA band shift and UV cross-linking assays, we identified two nuclear proteins (approximately 38- and 52-kDa) and two cytoplasmic proteins (approximately 50- and 74-kDa) that bind specifically to AU-rich inhibitory sequences. Deletion of the AU- or U-rich sequences in the HPV-1 late 3' UTR substantially reduced inhibitory activity, indicating that the cellular proteins we identified may be involved in inhibition of HPV-1 late gene expression in nondifferentiated epithelial cells.