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Comparative Study
. 2015 Aug:482:72-8.
doi: 10.1016/j.virol.2015.03.015. Epub 2015 Mar 30.

Immune evasion activities of accessory proteins Vpu, Nef and Vif are conserved in acute and chronic HIV-1 infection

Petra Mlcochova  1 , Luis Apolonia  2 , Silvia F Kluge  3 , Aishwarya Sridharan  1 , Frank Kirchhoff  3 , Michael H Malim  2 , Daniel Sauter  3 , Ravindra K Gupta  1
Affiliations
Comparative Study

"V体育ios版" Immune evasion activities of accessory proteins Vpu, Nef and Vif are conserved in acute and chronic HIV-1 infection

Petra Mlcochova et al. Virology. 2015 Aug.

Abstract

Heterosexual HIV-1 transmission has been identified as a genetic bottleneck and a single transmitted/founder (T/F) variant with reduced sensitivity to type I interferon initiates productive infection in most cases VSports手机版. We hypothesized that particularly active accessory protein(s) may confer T/F viruses with a selective advantage in establishing HIV infection. Thus, we tested vpu, vif and nef alleles from six T/F and six chronic (CC) viruses in assays for 9 immune evasion activities involving the counteraction of interferon-stimulated genes and modulation of ligands known to activate innate immune cells. All functions were highly conserved with no significant differences between T/F and CC viruses, suggesting that these accessory protein functions are important throughout the course of infection. .

Keywords: Accessory; Acute; Chronic; HIV; Nef; Vif; Vpu V体育安卓版. .

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"VSports最新版本" Figures

Fig. 1
Fig. 1
Vpu-mediated counteraction of tetherin, inhibition of NF-κB activation and downmodulation of CD4, NTB-A and CD1d. (A) Immunoblot showing expression of T/F and CC Vpu proteins in HEK293T cells transfected with pCG vpu IRES eGFP expression constructs. An antibody against AU-1 was used to detect C-terminally tagged Vpu. (B) HEK293T cells were co-transfected with expression vectors for HIV-1 Gag-Pol, VSV-G, a modified HIV genome encoding firefly luciferase, Vpu and tetherin or an empty vector control. 48 h post-transfection, viral supernatants were used to infect TZM-bl cells and luciferase activity was measured 24 h post-infection. Infectious virus yield in the presence of Vpu was normalized to that in the presence of empty vector (vector control). (C and D) HEK293T cells were co-transfected with Vpu expression plasmids, along with plasmid expressing an NF-κB-dependent firefly luciferase reporter construct and a pTAL promoter gaussia luciferase plasmid for normalization, and either a plasmid expressing (C) human tetherin or (D) a constitutively active mutant of IKKβ. 40 h post- transfection, dual luciferase assays were performed. Firefly luciferase signals were divided by the corresponding gaussia luciferase signals and normalized to the vector control. (E–J) HEK293T cells were transfected with (E and F) CD4 (G and H) NTB-A or (I and J) CD1d expression vectors and plasmids expressing eGFP alone or together with Vpu. Two days post-transfection CD4, NTB-A or CD1d surface expression was detected by fluorophore-conjugated antibodies and examined by flow cytometry. Mean fluorescence intensities were normalized to the control construct expressing only eGFP (vector control). Each data point represents one vpu allele. The mean of three independent experiments ± sd is shown. In all experiments, unpaired Student׳s t test was performed to examine differences between T/F and CC viruses. (E,G and I) Examples of primary experimental data from flow cytometry for one T/F and CC Vpu.
Fig. 2
Fig. 2
Vif-mediated counteraction of APOBEC3F and APOBEC3G. HEK293T cells were transfected with plasmids encoding NL4-3 Δvif, APOBEC3F or G and HA-tagged Vif proteins. 48 h post-transfection, producer cells and supernatants were harvested. (A) Expression of Vif proteins in producer cells was assessed by standard immunoblot. (B and C) Virion infectivity of supernatants generated in the presence of APOBEC3F (B) or APOBEC3G (C) was determined using TZM-bl indicator cells. Luciferase values were normalized to controls without Vif (vector control). The mean of four independent replicates ±sd is shown.
Fig. 3
Fig. 3
Nef-mediated downmodulation of CD4, CD28, MHC class I and enhancement of virion infectivity. (A) Immunoblot showing expression of T/F and CC Nef proteins in HEK293T cells transfected with pCG_nef IRES eGFP expression constructs. An antibody against AU-1 was used to detect C-terminally tagged Nef. (B–E) HEK293T cells were transfected with (B and C) CD4 or (D and E) CD28 expression vector and constructs expressing eGFP alone or together with Nef. Two days post-transfection CD4 or CD28 expression was detected by flow cytometry. Mean fluorescence intensities were normalized to the control construct expressing only eGFP (vector control). (F and G) MHC class I downregulation using HeLa.B27 cells transfected with constructs expressing eGFP alone or together with Nef. Two days post-transfection MHC I expression was analyzed by flow cytometry as described above. (H) Virion infectivity enhancement by Nef was measured using HEK293T cells co-transfected with HIV-1 NL4-3 and eGFP Nef. Supernatants were harvested 48 h post-transfection and p24-normalized virus was used to infect TZM-bl cells. Infectivity was normalized to the control construct expressing no nef allele (vector control). The mean of at least three independent experiments ±sd is shown. (B,D and F) Example of primary experimental data from flow cytometry for one T/F and CC Nef.
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