HIV-1 Gag recruits oligomeric Vpr via two binding sites in p6, but both mature p6 and Vpr are rapidly lost upon target cell entry

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The p12 region of MLV Gag and the p6 region of HIV-1 Gag contain late-domains required for virus budding. Additionally, the accessory protein Vpr is recruited into HIV particles via p6. Mature p12 is essential for early viral replication events, but the role of mature p6 in early replication is unknown. Using a proviral vector in which the and reading frames are uncoupled, we have performed the first alanine-scanning mutagenesis screens across p6, to probe its importance for early HIV-1 replication and to further understand its interaction with Vpr. The infectivity of our mutants suggests that, unlike p12, p6 is not important for early viral replication. Consistent with this, we observed that p6 is rapidly lost upon target cell entry in time-course immunoblotting experiments. By analysing Vpr incorporation in p6 mutant virions, we identified that the 15-FRFG-18 and 41-LXXLF-45 motifs previously identified as putative Vpr-binding sites are important for Vpr recruitment, but that the 34-ELY-36 motif also suggested to be a Vpr-binding site is dispensable. Additionally, disrupting Vpr oligomerization together with removing either binding motif in p6 reduced Vpr incorporation ∼25-50-fold more than inhibiting Vpr oligomerization alone and ∼10-25-fold more than deletion of each p6 motif alone, implying that multivalency/avidity is important for the interaction. Interestingly, using immunoblotting and immunofluorescence, we observed that most of Vpr is lost concomitantly with p6 during infection, but that a small fraction remains associated with the viral capsid for several hours. This has implications for the function of Vpr in early replication.
The p12 protein of MLV and the p6 protein of HIV-1 are both supplementary Gag cleavage products that carry proline-rich motifs which facilitate virus budding. Importantly, p12 has also been found to be essential for early viral replication events. However, whilst Vpr, the only accessory protein packaged into HIV-1 virions, is recruited via the p6 region of Gag, the function of both mature p6 and Vpr in early replication is unclear. Here, we have systematically mutated the p6 region of gag and have studied the effects on HIV infectivity and Vpr packaging. We have also investigated what happens to p6 and Vpr during early infection. We show that, unlike p12, mature p6 is not required for early replication and that most of the mature p6, and the Vpr that it recruits, are lost rapidly upon target cell entry. This has implications for the role of Vpr in target cells.

Journal details

Volume 95
Issue number 17
Pages e00554-21
Available online
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