A dileucine motif in HIV-1 Nef acts as an internalization signal for CD4 downregulation and binds the AP-1 clathrin adaptor
A dileucine motif in HIV-1 Nef acts as an internalization signal for CD4 downregulation and binds the AP-1 clathrin adaptor
Patricia A. Bresnahan*†‡, Wes Yonemoto*†, Sharon Ferrell*,
Debora Williams-Herman§, Romas Geleziunas* and Warner C. Greene*‡
Current Biology 1998, 8: 1235-1238
Summary
Human immunodeficiency virus 1 (HIV-1) Nef downregulates CD4 expression by increasing endocytosis of surface receptors and decreasing CD4 trafficking from the Golgi network to the plasma membrane. Although HIV-2 binds to AP-1 and AP-2 adaptors through terminal tyrosine-based motifs, these motifs are not found in HIV-1, and thus the mechanism by which Nef interacts with cellular signaling is unknown. In this paper, the authors investigate the role of a highly conserved dileucine motif in HIV-1 Nef that acts as an internalization signal and is required in CD4 downregulation. In addition, the authors provide evidence that supports dileucine motif-dependent binding of HIV-1 Nef to the AP-1 adaptor, as well as an evolutionary difference between HIV-1 and HIV-2 structural motifs for binding cellular adaptors.
Results
CD4 expression was characterized for human 293 cells after transfection with either HIV-1 NL4-3 Nef or mutant Nef variants. Flow cytometry was used to measure the percentage of cells that were CD4+, and immunoblotting was used to confirm Nef expression for each of the test groups. The groups transfected with Nef, namely + Nef, + Nef E160A, and + Nef P167A, showed low amounts of CD4 surface markers. On the other hand, the groups transfected with mutant Nef showed expression of CD4 surface markers comparable to the CD4 only group with no Nef transfection. Thus, this data confirms that Nef downregulates CD4 and that dysfunctional Nef will not downregulate CD4.
Expression of CD8 and CD8-Nef chimeras on human 293 cells was characterized using FACS. The top figure shows that the percentage of CD8-positive cells for the mutant Nef LL/AA was comparable to that of CD8 transfection only and much higher than that of wild-type Nef, which indicates that “CD8-Nef LL/AA mutant is preferentially expressed at the cell surface compared to CD8-Nef.” The middle figure shows that internalization of CD8-Nef occurred nearly four times faster than that of CD8-Nef LL/AA mutant, which shows that the dileucine motif does have an effect on CD8 internalization rate. The bottom figure shows that cell lysates had similar amounts of CD8-Nef and CD8-Nef LL/AA.
Shortfalls:
-Although the paper does extensive characterization using immunoprecipitation, FACS, flow cytometry, immunoblotting, and SDS-PAGE, additional characterization through other techniques such as qRT-PCR showing amount of expression for wild-type Nef and mutant Nef LL/AA, would have helped to support their findings on Nef’s HIV-1 effect of CD4 downregulation.
-The experiments had low sample sizes (N=2 or 3 in most cases), and more experiments could have been done in order to validate the mean and standard deviations for the percentage of CD4+ and CD8+ cells.
7 comments:
I agree with the suggestion of using qRT-PCR as it can also provide time data for the downregulation of CD4. An alternate way to achieve getting this time data could be tagging the cell-surface CD4 with an Fluorescent-conjugated antibody and detect signal intensity through FACS as well.
Does anyone know what the minimum acceptable number of samples for significance is for tests like these?
You mention that the paper discusses evolutionary differences between HIV-1 and HIV-2 structural motifs, but never allude to this notion in your results. It might be worth mentioning some of these findings.
I like your shortfalls section, though. Your points are very well thought-out.
@Phil:
It depends on the statistical test and level of confidence you want. The sample size can vary quite a bit depending on your statiscal power level (usually 0.8) and alpha level/p-value (usually 0.01 to 0.05).
For a p-value of 0.05 and power of 0.8, you actually need quite a few samples to claim statistical significance. According to this site, http://www.danielsoper.com/statcalc/calc47.aspx, you need 51 samples per group. Having that many samples for this kind of experiment is probably unfeasible, so I assume that's why they went with just N = 2 or 3.
@Matt:
I didn't include the data supporting evolutionary differences between HIV-1 and HIV-2 structural motifs due to the paper limit.
The paper provides data that shows that the dileucine motif is highly conserved in HIV-1 alleles, which suggests that the motif is a key structural element of Nef for HIV-1. For HIV-2, a leucine motif is well-conserved. Thus, HIV-1 and HIV-2 have developed differnt motifs that interact with cellular adaptors in different ways, most likely due to evolutionary pressures over time.
I noticed your paper was published in 1998. Have there been any recent developments or research further characterizing the role of Nef in HIV-1 or its motifs?
@Kal:
There have, actually. Since HIV-1 is a really hot topic, there have still been research papers published recently that investigate the Nef protein in greater detail, including effects of the dileucine motif and on adaptor protein-mediated pathways.
does the paper mention how are the surface markers on the CD4 t cell are analyzed? and how does the number of cd4 cells are quantitative analyzed ?
Post a Comment