Abstract
BackgroundThe human immunodeficiency virus type 1 (HIV-1) central DNA Flap is generated during reverse transcription as a result of (+) strand initiation at the central polypurine tract (cPPT) and termination after a ca. 100 bp strand displacement at the central termination sequence (CTS). The central DNA Flap is a determinant of HIV-1 nuclear import, however, neither cPPT nor CTS mutations entirely abolish nuclear import and infection. Therefore, to determine whether or not the DNA Flap is essential for HIV-1 nuclear import, we generated double mutant (DM) viruses, combining cPPT and CTS mutations to abolish DNA Flap formation.ResultsThe combination of cPPT and CTS mutations reduced the proportion of viruses forming the central DNA Flap at the end of reverse transcription and further decreased virus infectivity in one-cycle titration assays. The most affected DM viruses were unable to establish a spreading infection in the highly permissive MT4 cell line, nor in human primary peripheral blood mononuclear cells (PBMCs), indicating that the DNA Flap is required for virus replication. Surprisingly, we found that DM viruses still maintained residual nuclear import levels, amounting to 5-15% of wild-type virus, as assessed by viral DNA circle quantification. Alu-PCR quantification of integrated viral genome also indicated 5-10% residual integration levels compared to wild-type virus.ConclusionThis work establishes that the central DNA Flap is required for HIV-1 spreading infection but points to a residual DNA Flap independent nuclear import, whose functional significance remains unclear since it is not sufficient to support viral replication.
Highlights
The human immunodeficiency virus type 1 (HIV-1) central DNA Flap is generated during reverse transcription as a result of (+) strand initiation at the central polypurine tract and termination after a ca. 100 bp strand displacement at the central termination sequence (CTS)
We previously found that the two A-boxes have the most profound impact on HIV-1 infectivity when mutated, followed in importance by the G- and lastly the T-box
The addition of a single non-synonymous mutation has the most profound effect on viral replication [9]. This mutant introduces an amino acid change at position 188 of the integrase coding region (K188R), the control virus with the same amino acid change behaved like wild-type virus in terms of virus production and viral DNA synthesis [9]
Summary
The human immunodeficiency virus type 1 (HIV-1) central DNA Flap is generated during reverse transcription as a result of (+) strand initiation at the central polypurine tract (cPPT) and termination after a ca. 100 bp strand displacement at the central termination sequence (CTS). The human immunodeficiency virus type 1 (HIV-1) central DNA Flap is generated during reverse transcription as a result of (+) strand initiation at the central polypurine tract (cPPT) and termination after a ca. The central DNA Flap is a determinant of HIV-1 nuclear import, neither cPPT nor CTS mutations entirely abolish nuclear import and infection. The reverse transcriptase (RT) of retroviruses converts the (+) strand RNA genome into double-stranded DNA prior to nuclear import. The primer used for (-) strand synthesis is a cellular tRNA already present in the retroviral particles prior to infection and anneals to the 5’ region of the genome at the primer binding site (PBS). Similar to central (+) strand initiation, the termination of reverse transcription is a further feature that distinguishes lentiviruses from other orthoretroviruses. As a result of the cPPT and CTS cis-acting sequences, the final product of lentiviral reverse transcription contains a ca. 100 nt overlap, or “DNA Flap”, at the centre of the genome
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