Clearly different mechanisms of enhancement of short-lived Nef-mediated viral infectivity between SIV and HIV-1

Abstract

Background: One of the major functions of Nef is in the enhancement of the infectivity of the human and simian immunodeficiency viruses (HIV and SIV, respectively). However, the detailed mechanism of the enhancement of viral infectivity by Nef remains unclear. Additionally, studies of mechanisms by which Nef enhances the infectivity of SIV are not as intensive as those of HIV-1.

Methods: We generated short-lived Nef constructed by fusing Nef to a proteasome-mediated protein degradation sequence to characterize the Nef role in viral infectivity.

Results: The apparent expression level of the short-lived Nef was found to be extremely lower than that of the wild-type Nef. Moreover, the expression level of the short-lived Nef increased with the treatment with a proteasome inhibitor. The infectivity of HIV-1 with the short-lived Nef was significantly lower than that with the wild-type Nef. On the other hand, the short-lived Nef enhanced the infectivity of SIVmac239, an ability observed to be interestingly equivalent to that of the wild-type Nef. The short-lived Nef was not detected in SIVmac239, but the wild-type Nef was, suggesting that the incorporation of Nef into SIVmac239 is not important for the enhancement of SIVmac239 infectivity.

Conclusions: Altogether, the findings suggest that the mechanisms of infectivity enhancement by Nef are different between HIV-1 and SIVmac239. Lastly, we propose the following hypothesis: even when the expression level of a protein is extremely low, the protein may still be sufficiently functional.

Figure 1

Construction of short-lived Nef expression vector. Schematic representations of Nef-WT and Nef-CP used in this study (A). HEK293 cells were transfected with pNef_mac239-WT or pNef_mac239-CP. After a 48 h cultivation, HEK293 cells transfected with pNef_mac239-CP were treated with 20 μM MG132 for 0 or 6 h. The cells were collected and subjected to SDS-PAGE and western immunoblot analysis to detect Nef_mac239-WT and Nef_mac239-CP using the anti-V5 antibody. β-Actin was also detected using the anti-β-actin antibody, as described in Materials and Methods (B). The relative band intensities of Nef_mac239-CP and β-actin were quantified using Fujifilm Image Gauge software. The expression levels of Nef_mac239-CP relative to those of β-actin treated with and without MG132 were calculated using the obtained intensities and compared (C).

Figure 2

Difference in incorporation level in virions between Nef _mac239 -WT and Nef _mac239 -CP. pBRmac239Δnef/luc was cotransfected into HEK293 cells with either pNef_mac239-WT, pNef_mac239-CP, or an empty vector. For virion preparation, the supernatants were subjected to ultracentrifugation. Each viral pellet was then subjected to SDS-PAGE and western immunoblot analysis to detect Nef, Env, and p27, as described in Materials and Methods. The mock sample was electrophoresed on a lane distant from two lanes of the Nef_mac239-WT and Nef_mac239-CP.

Figure 3

Comparison of effects of Nef-WTs and Nef-CPs on enhancement of viral infectivity. pBRmac239Δnef/luc was cotransfected into HEK293 cells with either pNef_mac239-WT, pNef_mac239-CP, pNef_mac239-G2A, pNef_mac239-CP-G2A, or an empty vector (A). pBRmac239Δnef/luc was cotransfected into HEK293 cells with either pNef_JR-CSF-WT, pNef_JR-CSF-CP, or an empty vector (B). pNL-CHΔenvΔnef/luc and psvJR-FLenv were cotransfected into HEK293 cells with either pNef_JR-CSF-WT, pNef_JR-CSF-CP, or an empty vector (C). pNL-CHΔenvΔnef/luc and psvJR-FLenv were cotransfected into HEK293 cells with either pNef_mac239-WT, pNef_mac239-CP, or an empty vector (D). After a 72 h cultivation, the cells were collected and subjected to SDS-PAGE and western immunoblot analysis to detect Nef and β-actin, as described in Materials and Methods (top panels). The supernatants were subjected to SIV p27 or HIV-1 p24 enzyme-linked immunosorbent assay (ELISA) and infectivity assay using MAGIC-5 cells, as described in Materials and Methods (bottom panels). Each bar represents the mean standard deviation (n = 3).

Figure 4

Effect of low expression level of Nef _mac239 on SIV _mac239 infectivity. pBRmac239Δnef/luc was cotransfected into HEK293 cells with 1 μg of a mixture of pNef_mac239-WT and an empty vector, at amounts of pNef_mac239-WT in grams indicated. After a 72 h cultivation, the cells were collected and subjected to SDS-PAGE and western immunoblot analysis to detect Nef and β-actin, as described in Materials and Methods (A). The supernatants were subjected to SIV p27 ELISA and infectivity assay using MAGIC-5 cells as described in Materials and Methods (B). Each bar represents the mean standard deviation (n = 3).