Overlapping retrovirus U5 sequence elements are required for efficient integration and initiation of reverse transcription

Abstract

A secondary structure in the 5' noncoding region of avian retrovirus RNA, called the U5-leader stem, was shown previously to have a role in initiation of reverse transcription (D. Cobrinik, L. Soskey, and J. Leis, J. Virol. 62:3622-3630, 1988). We now show that an additional RNA secondary structure near the U5 terminus, called the U5-IR stem, is also important for reverse transcription. Mutations that disrupt the U5-IR stem cause a replication defect associated with both a decrease in synthesis of viral DNA in infected cells and a decrease in initiation of reverse transcription in melittin-permeabilized virions. Structure-compensating base substitutions in the U5-IR restore reverse transcription efficiency. In viral DNA, U5-IR sequences are included in the U5 term'inal region that functions as a viral integration donor site. When base substitutions are introduced into these sequences, a reduced efficiency of integration in vitro and in vivo is observed. These observations indicate that U5-IR sequences have a structural role in reverse transcription of viral RNA and a sequence-specific role in the integration of viral DNA.

Role of the open reading frames of Rous sarcoma virus leader RNA in translation and genome packaging.

“…The initiation and stop codons of the uORF1 and -2 are shown in bold characters; sequence substitutions of the mutants pAM1, pAM2 and pAM2130 are shown in boxes; the pADell sequence deletion is indicated by the bar. U5-IR and U5-leader are structures shown by Cobrinik et al (1991) to be involved in reverse transcription and in integration. The lowest calculated AG for this structure is -40 kcal/mol.…”

“…The uORFs 1 and 2 are localized in a region of the leader with a complex secondary structure (Cobrinik et al, 1988(Cobrinik et al, , 1991Aiyar et al, 1992;Hackett et al, 1992). This region, called the primer-binding loop (PBL)2,contains, in addition to the two first uORFs, the tRNA primer-binding site and two motifs involved in reverse trancription (Figure 7; U5-IR and U5-leader stem; Cobrinik et al, 1991). This structure appears to be conserved between different retrovirus species such as MMTV, MuLV, SNV and HIV (Cobrinik et al, 1988).…”

“…growth rate was also impaired (Figure 4). It is interesting to compare the properties of pAM2 with those of mutant S4C (see Figure IC in Cobrinik et al, 1991): the mutant S4C has two four-base complementary mutations in the U5-IR stem structure (maintaining the predicted secondary structure but altering its sequence, changing AUG2 to AUC). Like pAM2, S4C is impaired in its growth rate and shows no defect in viral RNA packaging.…”

“…The presence of smaller bands in Figure 6, in addition to the 101 base cDNA, probably represents premature termination of the reverse transcriptase under the conditions used (additional bands were consistently observed in the cDNA10 synthesis experiment). One possible explanation for the growth defect of pAM2 is an effect on integration, since mutations in the region around AUG2 have been shown to result in an integration defect, altering the DNA sequence recognized by the IN (integrase) protein (Cobrinik et al, 1991). 3752…”

A specific orientation of RNA secondary structures is required for initiation of reverse transcription

“…Details of initiation of DNA synthesis are not fully understood. However, the process is influenced by a series of viral 5' noncoding RNA secondary structures which are capable of modulating the efficiency of the reaction (1,7,8). These secondary structures are near the primer-binding site (PBS) and are called the U5-leader stem, the U5-IR stem, and the U5-TifiC interaction region.…”