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. 2012 May 25;427(1):67-75.
doi: 10.1016/j.virol.2012.02.002. Epub 2012 Mar 3.

HIV-2 viral protein X (Vpx) ubiquitination is dispensable for ubiquitin ligase interaction and effects on macrophage infection

Anna McCulley  1 , Lee Ratner
Affiliations

HIV-2 viral protein X (Vpx) ubiquitination is dispensable for ubiquitin ligase interaction and effects on macrophage infection

Anna McCulley et al. Virology. .

Abstract

HIV-2 Vpx, a virus-associated accessory protein, is critical for infection of non-dividing myeloid cells. To understand the function of Vpx ubiquitination, interaction with an E3 ubiquitin ligase complex, and ability to overcome an inhibition of reverse transcription, we analyzed Vpx lysine mutants for their function and replication capability in macrophages. Both Wt Vpx and Vpx TA (lysine-less Vpx) localized to the cytoplasm and nucleus in HeLa cells. All HIV-2 Vpx lysine mutants were functional in virion packaging. However, ubiquitination was absent with Vpx TA and Vpx K84A mutants, indicating a lack of ubiquitin on positions K68 and K77. Mutants Vpx K68A and K77A were unable to infect macrophages due to impaired reverse transcription from loss of interaction with the ubiquitin substrate receptor, DCAF1. Even though Vpx K84A lacked ubiquitination, it bound DCAF1, and infected macrophages comparable to Wt Vpx.

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Figures

Figure 1
Figure 1. Schematic representation of the 6xHis-tagged HIV-2 Vpx constructs and their expression
Schematic depiction of Vpx lysine residues that were individually substituted for alanine at positions 68, 77, 84 and in triple combination, TA (triple alanine), in which case every lysine in Vpx is substituted. All constructs were N-terminally labeled with 6xHis tag. Only partial amino acid sequences are shown for Vpx. Western blot showing the expression of the 6xHis-tagged Vpx mutants. HEK 293T cells were transfected with mock, 6xHis-tagged Vpx wild-type (Wt), 6xHis-tagged Vpx triple alanine (TA), 6xHis-tagged Vpx K68A, 6xHis-tagged Vpx K77A, and 6xHis-tagged Vpx K84A constructs. Forty eight hours post-transfection, cells were lysed and proteins were resolved by SDS-PAGE. Blots were analyzed for Vpx expression using an anti-Vpx mAb.
Figure 2
Figure 2. Vpx exhibits a perinuclear localization pattern in HeLa cells
The 6xHis-tagged Vpx Wt and 6xHis-tagged Vpx TA constructs were transfected into HeLa cells cultured on coverslips. The cells were fixed, permeabilized, and stained with anti-Vpx and anti-Nup98. Secondary antibodies included Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 562-conjugated goat anti-rabbit IgG. Nuclei were visualized with TO-PRO3 stain. Confocal microscopy was used to obtain images at 24h post-transfection.
Figure 3
Figure 3. Viral incorporation of HIV-2 Vpx Wt and Vpx lysine mutants
Immunoblot analysis of Vpx mutant proteins in 293T cellular lysates and virions. HEK 293T cells were co-transfected with proviral clone pES or pESdelVpx (which lacks Vpx expression) and 6xHis-tagged Vpx Wt or mutant constructs. On day three post-transfection, supernatants were clarified through a 0.22 μm filter, and virus concentrated on a 20% sucrose cushion. Cellular and viral lysates were resolved by SDS-PAGE. Both, virus and cell lysates were analyzed by Western blot with an anti-Vpx mAb and HIV-1 antiserum against Gag. Band intensity was determined by imaging (BioRad Image Lab 3.0). Relative intensity was normalized to that of capsid (CA) levels, relative to those of pESdelX+Wt. Relative density is reported as ratios of supernatant to cellular lysates.
Figure 4
Figure 4. Effect of Vpx lysine substitution on ubiquitination of Vpx
(A) HEK 293T cells were transfected with 6xHis-tagged Vpx construct and pcDNA (mock). Three days post-transfection, the cells were lysed, and the proteins resolved by SDS-PAGE and analyzed by Western blot using an anti-Vpx mAb. (B) Assessment of ubiquitination of 6xHis-tagged Vpx lysine mutants. 293T cells were transfected with 6xHis-tagged Vpx and 6xHis-tagged Vpx lysine mutant constructs in the presence or absence of plasmid that encodes the Flag-tagged ubiquitin K48R. Three days post-transfection, the cells were lysed and purified using a Ni-NTA pulldown of 6XHis-tagged Vpx. Lysates were resolved by SDS-PAGE, and analyzed by blotting for ubiquitinated Vpx with anti-Flag mAb. Lower panel represents a second independent assessment for ubiquitinated Vpx. C) Endogenous ubiquitination and Vpx expression (Post-pulldown) was detected by re-blotting the Ni-NTA pulldown blot with anti-Vpx mAb. An aliquot of lysates was saved before exposure to Ni-NTA beads and was blotted with the anti-Vpx mAb (Pre-pulldown). Post-pulldown, Pre-pulldown, and endogenous ubiquitin blot had different exposure time.
Figure 5
Figure 5. Alkaline treatment of Vpx
(A) HEK 293T cells were transfected with 6xHis-tagged Vpx construct and pcDNA (mock). Three days post-transfection, the cells were lysed in denaturing conditions, and Vpx was purified using Ni-NTA beads. After several washes with lysis buffer, Vpx bound to beads was divided into two samples. One sample was treated with PBS (control) and the other sample was treated with 0.1M NaOH. Both parts were incubated for 2h at 37°C. Proteins were resolved by SDS-PAGE and analyzed by Western blot using an anti-Flag mAb. (B) Endogenous ubiquitination was detected by re-blotting the PBS and NaOH treated blot with anti-Vpx mAb.
Figure 6
Figure 6. Effect of Vpx lysine substitutions on viral infectivity
Viral DNA synthesis in human MDMs. The VSV-g pseudotyped cell-free virus was prepared in 293T cells that were co-transfected with proviral construct pES, and pESdelX (lacks expression of Vpx), and 6xHis-tagged Vpx mutant constructs and pVSV-g. The MDMs were mock infected, infected with viruses expressed from pES, or pESdelX (which lacks Vpx expression), or viruses expressed from pESdelX trans-complemented with 6xHis-tagged Vpx lysine mutants. Equal amounts of virus, which was titered on TMZ-bl reporter cells, were used in the infection assays, and all viral samples were treated with DNase I before infection of MDMs. AZT (10 μM) was added as a control 24h and 6h prior to infection to specified wells. Cellular DNA was extracted at 24 h and 48 h post-infection and subjected to real-time PCR analysis of early and late reverse transcription products. The data are an average of two independent experiments with error bars representing the standard deviation of the two samples.
Figure 7
Figure 7. Vpx lysine substitutions affect the interaction of Vpx with DCAF
HEK 293T cells were transfected with mock, 6xHis-tagged Vpx Wt, 6xHis-tagged Vpx TA, 6xHis-tagged Vpx K68A, 6xHis-tagged Vpx K77A, and 6xHis-tagged Vpx K84A. Forty eight hours post-transfection, the cells were lysed, and Vpx was immunoprecipitated using anti-Vpx mAb and Protein A agarose beads. Lysates were resolved with SDS-PAGE and immunoblotted for DCAF1 and Vpx.
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