The Major Human Immunodeficiency Virus Type 2 (HIV-2) Packaging Signal Is Present on All HIV-2 RNA Species: Cotranslational RNA Encapsidation and Limitation of Gag Protein Confer Specificity

Abstract

Deletion of a region of the human immunodeficiency virus type 2 (HIV-2) 5 leader RNA reduces genomic RNA encapsidation to about 5% that of wild-type virus with no defect in viral protein production but severely limits virus spread in Jurkat T cells, indicating that this region contains a major cis-acting encapsidation signal, or psi (⌿). Being upstream of the major splice donor, it is present on all viral transcripts. We have shown that HIV-2 selects its genomic RNA for encapsidation cotranslationally, rendering wild-type HIV-2 unable to encapsidate vector RNAs in trans . Virus with ⌿ deleted, however, encapsidates an HIV-2 vector, demonstrating competition for Gag protein. HIV-2 overcomes the lack of packaging signal location specificity by two novel mechanisms, cotranslational packaging and competition for limiting Gag polyprotein.

The packaging signal of simian immunodeficiency virus is upstream of the major splice donor at a distance from the RNA cap site similar to that of human immunodeficiency virus types 1 and 2

“…Some groups have found packaging signals in positions analogous to those in HIV-1 (Garzino Demo et al, 1995;Sadaie et al, 1998). We have found consistently only a minor effect on packaging attributable to this region (McCann & Lever, 1997;Griffin et al, 2001;Kaye & Lever, 1999). Most groups agree that there is some contribution to packaging from regions upstream of the SD and we have, on repeated experiments, found this region to be the dominant packaging signal.…”

“…For lentiviruses, regulatory and accessory proteins are encoded on multiply spliced mRNAs that are translated on free cytoplasmic ribosomes, where they could conceivably compete with the genomic RNA for packaging. For HIV-2, specificity is maintained by cotranslational packaging of the Gagencoding (Kaye & Lever, 1999;Griffin et al, 2001) (unspliced) message and apparent limiting availability of Gag available to capture other RNAs (Griffin et al, 2001). Given the genetic relatedness of the primate lentiviruses, it was of interest to see where the encapsidation signal of simian immunodeficiency virus (SIV) would be found.…”

Elements Located Upstream and Downstream of the Major Splice Donor Site Influence the Ability of HIV-2 Leader RNA To Dimerize in Vitro

“…Experiments from the Lever group showed that HIV-2 selects its genomic RNA for encapsidation cotranslationally (20). Two elements seem to mediate this activity: the nucleocapsid domain of the Gag protein and the 380-408 sequence located just upstream of the SL1 sequence in the leader RNA region, called the encapsidation signal or Ψ (Figure 8) (20). Mutagenesis studies have shown that retroviral encapsidation and dimerization elements overlap in vivo (for a review, see (40)).…”

“…The gag initiation codon is indicated by the solid horizontal bar below the sequence. ψ represents the core encapsidation signal of HIV-2 genomic RNA (nucleotides 380 to 408) defined by deletion mutagenesis (20). Thick lines indicate the PBS and the entire SL1 sequence (nucleotides 409 to 439; (29)).…”

“…We also tested the influence of the nucleocapsid protein on the formation of tight dimer with 1-444 and 1-561 RNAs and observed that, similarly to the HIV-1 system, the yield of SL1-mediated tight dimerization increased in the presence of nucleocapsid protein, even with the large HIV-2 leader RNA fragments that were unable to form tight dimers without protein. Since the SL1 structure is very close to the core encapsidation signal of HIV-2 RNA (20) and one of the dimer-interfering elements encompasses the gag initiation codon, our results suggest a model of how the processes of translation, dimerization, and encapsidation of HIV-2 RNA and proteins could be interdependent in vivo.…”