Nef proteins from simian immunodeficiency virus-infected chimpanzees interact with p21-activated kinase 2 and modulate cell surface expression of various human receptors

Abstract

The accessory Nef protein allows human immunodeficiency virus type 1 (HIV-1) to persist at high levels and to cause AIDS in infected humans. The function of HIV-1 group M subtype B nef alleles has been extensively studied, and a variety of in vitro activities believed to be important for viral pathogenesis have been established. However, the function of nef alleles derived from naturally simian immunodeficiency virus (SIV)-infected chimpanzees, the original host of HIV-1, or from the HIV-1 N and O groups resulting from independent zoonotic transmissions remains to be investigated. In the present study we demonstrate that SIVcpz and HIV-1 group N or O nef alleles down-modulate CD4, CD28, and class I or II MHC molecules and up-regulate surface expression of the invariant chain (Ii) associated with immature major histocompatibility complex (MHC) class II. Furthermore, the ability of Nef to interact with the p21-activated kinase 2 was generally conserved. The functional activity of HIV-1 group N and O nef genes did not differ significantly from group M nef alleles. However, SIVcpz nef genes as a group showed a 1.8- and 2.0-fold-higher activity in modulating CD28 (P = 0.0002) and Ii (P = 0.016) surface expression, respectively, but were 1.7-fold less active in down-regulating MHC class II molecules (P = 0.006) compared to HIV-1 M nef genes. Our finding that primary SIVcpz nef alleles derived from naturally infected chimpanzees modulate the surface expression of various human cellular receptors involved in T-cell activation and antigen presentation suggests that functional nef genes helped the chimpanzee virus to persist efficiently in infected humans immediately after zoonotic transmission.

FIG. 1.

Evolutionary relationships among HIV-1 and SIVcpz Nef sequences. A phylogenetic tree was constructed based on Nef amino acid sequences by the neighbor-joining method. The tree was rooted with the SIVgsn Nef sequence as an out-group. Confidence in tree nodes was determined with 1,000 bootstrap resampling replicates in parallel with neighbor joining and maximum parsimony (confidence values before and after the slash, respectively).

FIG. 2.

Alignment of HIV-1 and SIVcpz Nef sequences. The NL4-3 sequence is shown in the upper panel for comparison. Some conserved sequence elements in Nef, the position of the polypurine tract (PPT), and the start of the 3′ long terminal repeat (LTR) are indicated schematically. Asterisks above the alignment indicate positions where amino acid variations between the different groups are observed; the number gives the corresponding amino acid position in the NL4-3 Nef. Dots indicate identity with the consensus sequence, and dashes indicate gaps introduced to optimize the alignment. bdg, binding; VIH, catalytic subunit of vacuolar ATPase.

FIG. 3.

Interaction of HIV-1 and SIVcpz Nef proteins with PAK-2. 293T cells were cotransfected with expression plasmids for HA-tagged PAK-2, Cdc42V12, and the AU1-tagged Nef proteins indicated. An aliquot of the cell lysates was directly separated by SDS-PAGE and analyzed for Nef and PAK-2 expression by Western blot analysis with a mixture of AU-1- and HA-specific antibodies (upper panel). To detect PAK-2 interaction, Nef immunocomplexes were precipitated with anti-AU1 antibody, subjected to IVKAs, and separated by SDS-PAGE (lower panel).

FIG. 4.

Modulation of human cell surface receptors by nef alleles derived from the HIV-1 M, N, and O groups or SIVcpz(P.t.t.) and SIVcpz(P.t.s.) groups. Jurkat T or HeLa-CIITA cells were transfected with the indicated HIV-1 and SIVcpz Nef expression plasmids and assayed for surface expression of CD4, CD28, MHC-I, MHC-II, and Ii. Quantification was performed as described in Materials and Methods. In the upper three panels, the areas of no, low, medium, and high levels of GFP and, hence, Nef expression (72) are indicated. The results were confirmed in two independent experiments.

FIG. 5.

Functional activity of nef alleles derived from different groups of HIV-1 and SIVcpz. For the quantitation of Nef-mediated CD4, CD28, Ii, and MHC-I or -II downregulation, the mean channel numbers of red fluorescence were determined for cells with high levels of GFP and, hence, Nef (72). The numbers obtained for cells transfected with vector expressing GFP only were divided by the corresponding numbers obtained for cells coexpressing Nef and GFP, to calculate the values for down- or up-modulation (n-fold), respectively. Each symbol represents the functional activity of one individual nef allele. The results were confirmed in at least three independent experiments. The horizontal bars indicate average activities. The open triangle in the HIV-1 O panel specifies the Ca9k7 nef allele which contains an E/DXXXLL to VXXXLL mutation in the C-proximal endocytosis signal. The open symbols in the SIVcpz panels specify the three Cam nef alleles that have been obtained after short-term passage of SIVcpz in human PBMC. Please note that different scales are used on the y axes.