Functional conservation and coherence of HIV-1 subtype A Vpu alleles

Abstract

Functional studies of HIV-1 proteins are normally conducted using lab adapted strains of HIV-1. The extent of those functions in clinical strains is sometimes unknown. In this study, we amplified and sequenced HIV-1 Vpu from 10 Iranian patients infected with HIV-1. Phylogenetic analysis indicated that the Vpu alleles were closely related to the CRF35_AD from Iran and subtype A Vpu. We addressed some of the well-established functions of the HIV-1 Vpu, as well as some of its recently reported functions. Ability of the clinical strains of subtype A Vpu alleles for downregulation of CD4 was similar to that of the lab adapted NL4.3 Vpu. Majority of the subtype A Vpu alleles performed stronger than NL4.3 Vpu for downregulation of SNAT1. The Vpu alleles differentially induced downregulation of HLA-C, ranging from no effect to 88% downregulation of surface HLA-C. Downregulation of tetherin and enhancement of virus release was similar for the subtype A Vpu alleles and NL4.3. Subtype A Vpu alleles were more potent when compared with NL4.3 for inhibition of NF-κB activation. Our study shows that subtype A Vpu alleles exert the classical functions of HIV-1 Vpu.

Figure 1. Phylogenetic analysis of HIV-1 isolates.

(A) DNA sequences of the Vpu alleles were aligned with Vpu of HIV-1 group M reference sequences. A phylogenetic tree was constructed and rooted using Vpu sequences of HIV-1 group N and SIVcpz. Ten Vpu alleles of the clinical isolates are shown with JU (Jundishapur University) suffixed by their respective clone number (1 to 10). For clarity, our isolates are indicated with filled circles (•). Gene accession numbers are indicated in brackets and the branch lengths represent the sequence dissimilarities. (B) A phylogenetic tree was constructed with partial Pol DNA sequences of the HIV-1 isolates and the reference sequences of HIV-1 group M. The tree was rooted with HIV-1 group N and SIVcpz. (C) Alignment of the deduced Vpu amino acid sequences from 10 Iranian isolates along with the NL4.3 Vpu. Dots represent identity to the sequence at the bottom and dashes represent gaps. Transmembrane and α-helical domains of Vpu are shaded gray.

Figure 2. Downregulation of cellular proteins by HIV-1 Vpu alleles.

(A) CEM-CD4⁺ cells were transduced with GFP-marked lentiviral vectors expressing Vpu alleles. A mutant form of Vpu (Vpu-S52, 56 N), incapable of downregulating CD4, was used as control. After 48 h, transduced cells were sorted using the GFP signal and subjected to Western blot to assess CD4 expression level by Vpu alleles (n = 3). (B) CEM-CD4⁺ cells were transduced with GFP-marked lentiviral vectors expressing Vpu alleles. GFP-positive cells were sorted after 48 h and analyzed using Western blot to assess downregulation of SNAT1 by Vpu alleles (n = 3). Asterisks indicate significant differences of subtype A Vpu alleles compared with NL4.3 Vpu. (C) Vpu alleles were tested for their ability to downregulates HLA-C on the cell surface of CEM-CD4⁺ cells transduced with GFP-marked lentiviral vectors. The bar graph shows summary of 3 independent experiments. Asterisks indicate significant differences of subtype A Vpu compared with NL4.3 Vpu.

Figure 3. Downregulation of surface tetherin and enhancing virus release by Vpu alleles.

(A) CEM-CD4⁺ cells were transduced with GFP-marked lentiviral vectors for expression of Vpu alleles. After 48 h, surface expression of tetherin was measured in the GFP-positive transduced cells (n = 3). (B) HeLa cells were cotransfected with HIV-1 ∆Vpu/Nef and expression vectors for expression of Vpu alleles. After 48 h, cell lysates and supernatants were analyzed using Western blot. Fold increase in virus release was calculated by quantifying the signal density of cell-free p24 in the supernatant. (C) HeLa cells were cotransfected with HIV-1 ∆Vpu/Nef and expression vectors for expression of the WT Vpu (NL4.3) or the mutant Vpu (S52, 56 N). Cell lysates and supernatants were analyzed as described for B.

Figure 4. Inhibition of NF-κB by Vpu alleles.

HEK293T cells were cotransfected with a vector for expression of MAVS as inducer of NF-κB and a firefly luciferase reporter construct controlled by five NF-κB binding sites. After 12 h, cells were transduced with lentiviral vectors for expression of Vpu alleles or empty vector as control (n = 3). Luciferase activities were measured 36 h post-transduction. Luciferase activity of the empty vector in the presence of the MAVS inducer was set on 100% as the control. Luciferase activities of other samples were calculated based on the control.