Randomization and In Vivo Selection Reveal a GGRG Motif Essential for Packaging Human Immunodeficiency Virus Type 2 RNA

Abstract

The packaging signal () of human immunodeficiency virus type 2 (HIV-2) is present in the 5 noncoding region of RNA and contains a 10-nucleotide palindrome (pal; 5-392-GGAGUGCUCC) located upstream of the dimerization signal stem-loop 1 (SL1). pal has been shown to be functionally important in vitro and in vivo. We previously showed that the 3 side of pal (GCUCC-3) is involved in base-pairing interactions with a sequence downstream of SL1 to make an extended SL1, which is important for replication in vivo and the regulation of dimerization in vitro. However, the role of the 5 side of pal (5-GGAGU) was less clear. Here, we characterized this role using an in vivo SELEX approach. We produced a population of HIV-2 DNA genomes with random sequences within the 5 side of pal and transfected these into COS-7 cells. Viruses from COS-7 cells were used to infect C8166 permissive cells. After several weeks of serial passage in C8166 cells, surviving viruses were sequenced. On the 5 side of pal there was a striking convergence toward a GGRGN consensus sequence. Individual clones with consensus and nonconsensus sequences were tested in infectivity and packaging assays. Analysis of individuals that diverged from the consensus sequence showed normal viral RNA and protein synthesis but had replication defects and impaired RNA packaging. These findings clearly indicate that the GGRG motif is essential for viral replication and genomic RNA packaging.The 5Ј leader RNA of retroviruses is involved in the regulation of many essential steps of the retroviral replication cycle. These steps include transcription, splicing, translation, genomic RNA dimerization, packaging, and reverse transcription (RT) (1, 8, 12, 27, 34-36, 38, 40). RNA packaging is a critical step during which two molecules of full-length genomic RNA are encapsidated into the budding virion in the form of a dimer (for a review, see reference 42). Furthermore, packaging is a highly selective and specific process in which unspliced genomic RNA is preferentially incorporated in viral particles over a vast excess of cellular mRNAs and spliced viral RNAs (31,32). This selectivity occurs through the interaction between trans-acting Gag polyprotein and cis-acting elements in the genomic RNA. The cis elements are known as packaging signals (psi or ⌿) and have been mapped in the 5Ј leader region of unspliced genomic RNA in many retroviruses (22,31,42).

HIV-2 Genome Dimerization Is Required for the Correct Processing of Gag: a Second-Site Reversion in Matrix Can Restore Both Processes in Dimerization-Impaired Mutant Viruses

“…The 3= end of pal (GCUCC-3=) is required for the formation of stem B and the extended SL-1. However, infectivity and viral replication are significantly impaired by the sole mutation of the 5= end of pal (5=-GGAGU), which is not part of stem B (5,41). Therefore, the precise sequence and structural elements of SL-1 required for HIV-2 genome dimerization, packaging, and viral replication remain incompletely defined.…”

Viral SELEX reveals individual and cooperative roles of the C-box and G-box in HIV-2 replication

“…The pSCR2, pAUG1, pCboxg, and pGboxc plasmids harbor deleterious mutations in the encapsidation signal (Baig et al 2009), the gag initiation codon, the C-box region , and the G-box region (541-CCACC-545), respectively.…”

“…To generate the proviral DNA libraries, we used a single-step ligation-independent DNA cloning protocol (In-Fusion; Clontech) as described previously (Baig et al 2009). Both the pSCR2AfeID and pAUG1AfeID vectors were prepared by an AfeI and XhoI digestion, followed by gel electrophoresis and gel extraction (5PRIME).…”

“…Several previous studies suggested that both the presence of the C-box and the G-box and their ability to base-pair affected dimerization in HIV-2 (Dirac et al 2002;Lanchy et al 2003aLanchy et al ,b, 2004Baig et al 2009) and in HIV-1 (Abbink and Berkhout 2003;Damgaard et al 2004;Abbink et al 2005;Song et al 2008). It was postulated that the C-box-G-box interaction, termed ''CGI'' in HIV-2 (Baig et al 2008) or ''U5-AUG duplex'' in HIV-1 (Abbink and Berkhout 2003), could modulate translation through occlusion of the gag start codon encompassed by the G-box (Abbink and Berkhout 2003;Lanchy et al 2003a,b;Damgaard et al 2004).…”

“…Here we used a viral SELEX technique (Berkhout and Klaver 1993;Baig et al 2009) to generate viable virus variants at the C-box or the G-box. When either of these sequences was randomized alone, wild-type-like sequences rapidly emerged as the dominant species.…”