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. 2005 Aug;79(16):10547-60.
doi: 10.1128/JVI.79.16.10547-10560.2005.

Primary sooty mangabey simian immunodeficiency virus and human immunodeficiency virus type 2 nef alleles modulate cell surface expression of various human receptors and enhance viral infectivity and replication

Jan Münch  1 , Michael Schindler, Steffen Wildum, Elke Rücker, Nicola Bailer, Volker Knoop, Francis J Novembre, Frank Kirchhoff
Affiliations

Primary sooty mangabey simian immunodeficiency virus and human immunodeficiency virus type 2 nef alleles modulate cell surface expression of various human receptors and enhance viral infectivity and replication

Jan Münch et al. J Virol. 2005 Aug.

Abstract

The nef gene of the pathogenic simian immunodeficiency virus (SIV) mac239 clone has been well characterized. Little is known, however, about the function of nef alleles derived from naturally SIVsm-infected sooty mangabeys (Cercocebus atys) and from human immunodeficiency virus type 2 (HIV-2)-infected individuals. Addressing this, we demonstrate that, similarly to the SIVmac239 nef, primary SIVsm and HIV-2 nef alleles down-modulate cell surface expression of human CD4, CD28, CD3, and class I or II major histocompatibility complex (MHC-I or MHC-II, respectively) molecules, up-regulate surface expression of the invariant chain (Ii) associated with immature MHC-II, inhibit early T-cell activation events, and enhance virion infectivity. Both also stimulate viral replication, although HIV-2 nef alleles were less active in this assay than SIVsm nef alleles. Mutational analysis showed that a dileucine-based sorting motif in the C-proximal loop of SIV or HIV-2 Nef is critical for its effects on CD4, CD28, and Ii but dispensable for down-regulation of CD3, MHC-I, and MHC-II. The C terminus of SIV and HIV-2 Nef was exclusively required for down-modulation of MHC-I, further demonstrating that analogous functions are mediated by different domains in Nef proteins derived from different groups of primate lentiviruses. Our results demonstrate that none of the eight Nef functions investigated had been newly acquired after cross-species transmission of SIVsm from naturally infected mangabeys to humans or macaques. Notably, HIV-2 and SIVsm nef alleles efficiently down-modulate CD3 and C28 surface expression and inhibit T-cell activation more efficiently than HIV-1 nef alleles. These differences in Nef function might contribute to the relatively low levels of immune activation observed in HIV-2-infected human individuals.

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Figures

FIG. 1.
FIG. 1.
Evolutionary relationships among HIV-2, SIVsm, and SIVmac Nef sequences. A phylogenetic tree was constructed using the minimum evolution criterion as implemented in MEGA3 (33). Bootstrap node support was calculated from 10,000 replicates. Nefs that were functionally characterized in this study are labeled with an asterisk.
FIG. 2.
FIG. 2.
Alignment of SIVmac239, SIVsm, and HIV-2 Nef sequences. The SIVmac239 sequence is shown in the upper row for comparison. Some conserved sequence elements in Nef, including the N-terminal myristoylation signal, N-proximal tyrosines (6, 8), PA residues known to be critical for CD4 down-modulation by 239wt Nef (26), the acidic and proline-rich regions, a diarginine motif, a C-proximal adaptor protein (AP) interaction site (10), a diacidic putative V1H binding site, and a Y residue involved in MHC-I down-regulation (39, 58), are indicated schematically. Dots indicate identity with the 239wt Nef sequence, and dashes indicate gaps introduced to optimize the alignment. The asterisk denotes the position of the premature stop codon in the HIV-2 310072 Nef. For comparison, the predicted amino acid sequence after the stop signal is shown in the alignment.
FIG. 3.
FIG. 3.
Expression of SIVsm and HIV-2 Nefs. (A) 293T cells were transfected with expression plasmids encoding the indicated AU1-tagged Nef proteins and GFP. Mock-transfected cells and a vector containing a disrupted nef gene (GFP only) were used as negative controls. GFP was detected to control for transfection efficiency and β-actin for total quantity of cellular protein. (B) Jurkat cells were transfected with a bicistronic vector expressing GFP and Nef or GFP alone. As described in Materials and Methods, cells were permeabilized, and Nef and GFP expression were detected simultaneously. Ranges for green fluorescence on cells defined as expressing no (N) GFP and low (L) and medium (M) levels of GFP are indicated. Compared to normal transfected Jurkat T cells, permeabilized cells contain smaller quantities of GFP. Therefore, the number of cells expressing high levels of GFP was relatively low and not used for quantitative analysis. The right panel shows the mean channel numbers of red Nef fluorescence for the three different regions of GFP expression. The results were confirmed in an independent experiment using different plasmid DNA preparations.
FIG. 4.
FIG. 4.
Modulation of human cell surface receptors by SIVsm and HIV-2 nef alleles. Jurkat T (rows A to D) or HeLa CIITA (rows E and F) cells were transfected with bicistronic vectors coexpressing the indicated nef alleles and GFP and assayed for surface expression of CD4, CD3, CD28, MHC-I, MHC-II, and Ii. The results were confirmed in two independent experiments and using the AU1-tagged Nef proteins.
FIG. 5.
FIG. 5.
HIV-2 and SIVsm nef alleles inhibit CD69 induction more severely than HIV-1 nef alleles. (A) Jurkat cells were either mock transfected (panels 1 and 2), transfected with a plasmid expressing GFP alone (panel 3), or transfected with plasmids expressing GFP together with the 239wt, FFm1, BEN, and NA7 nef alleles (panels 4 to 7) and cultured overnight in the absence (panel 1) or presence (panels 2 to 7) of PHA and stained for CD69 expression. Ranges for green fluorescence on cells defined as expressing no GFP (N) and low (L), medium (M), or high (H) levels of GFP are indicated in panels 3 to 7. Values give mean fluorescence intensity. Similar results were obtained in two independent experiments. (B) Inhibition of CD69 induction by the indicated nef alleles. CD69 expression levels on cells coexpressing different levels of GFP and Nef are shown relative to those measured on Jurkat T cells transfected with the control construct expressing only GFP. (C) Correlation between inhibition of PHA- and CD3-induced CD69 expression. Calculations were performed for Jurkat T cells expressing medium levels of GFP and the indicated nef alleles. Average values derived from three independent experiments are shown in panels B and C.
FIG. 6.
FIG. 6.
The dileucine-based sorting motif in HIV-2 or SIVmac Nef is not required for down-modulation of CD3, MHC-I, and MHC-II surface expression. Cells were transfected with constructs expressing either the wild-type NA7, 239, BEN, or FFm1 Nefs or mutant forms containing changes of EXXXL to AXXXA in the C-proximal adaptor protein interaction site. Assays were performed as described in Materials and Methods and confirmed in two independent experiments.
FIG. 7.
FIG. 7.
The C terminus of HIV-2 Nef is critical for MHC-I down-modulation but not for other Nef functions. (A) Mutations introduced into HIV-2 Nef. (B) The functional activity of the indicated Nef mutants was determined as described in the legend to Fig. 4. The NA7 AXXA Nef contains the changes P72A and P75A. The mutant SIVmac239 Nefs have been described previously (34, 58). Similar results were obtained in two independent experiments.
FIG. 8.
FIG. 8.
SIVsm and HIV-2 nef alleles enhance viral infectivity. (A) Generation of proviral SIVmac239 constructs carrying SIVsm and HIV-2 nef alleles. As described previously (31), the original SIVmac239 nef initiation codon and a second ATG at amino acid position 7 of the nef ORF were mutated to eliminate the overlap between the env and nef open reading frames. The various nef genes were cloned into the unique SacI and MluI restriction sites. (B) Infectivity of SIVmac239 without overlapping env-nef sequences. TZM-bl cells were infected in triplicate with the parental 239wt or 239nef* viruses or with mutant forms containing an intact (SSIV239nef+) or a prematurely truncated (SSIV239nef*) nef gene inserted downstream of env and upstream of a truncated U3 region as shown in panel A. (C) Infectivity of SIVmac239 variants containing the indicated HIV-1, SIVsm, or HIV-2 nef alleles. TZM-bl indicator cells were infected in triplicate with 293T cell-derived virus stocks containing 200 pg p27. Infectivity is shown relative to the recombinant virus containing the 239wt nef allele. Similar results were obtained in two independent experiments and using P4-CCR5 indicator cells.
FIG. 9.
FIG. 9.
Enhancement of viral replication by (A) SIVsm and (B) HIV-2 nef alleles. Replication kinetics of recombinant SIVmac239 variants containing the indicated SIVsm or HIV-2 nef alleles in 221-89 cells in the presence (left panels) or absence (right panels) of exogenous IL-2 are shown. Infections were performed using virus stocks containing 10 pg p27 antigen. All infections were performed at the same time and the 239wt and nef* controls are shown in both panels for comparison. Reverse transcriptase activity was determined using a phosphorimager. PSL, photon-stimulated light emission. Symbols: ▪, 239wt; □, nef*; •, K6 nef alleles; ×, uninfected cells. Similar results were obtained in two independent experiments.
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References

    1. Alexander, L., Z. Du, M. Rosenzweig, J. U. Jung, and R. C. Desrosiers. 1997. A role for natural simian immunodeficiency virus and human immunodeficiency virus type 1 Nef alleles in lymphocyte activation. J. Virol. 71:6094-6099. - PMC - PubMed
    1. Arganaraz, E. R., M. Schindler, F. Kirchhoff, M. J. Cortes, and J. Lama. 2003. Enhanced CD4 down-modulation by late stage HIV-1 nef alleles is associated with increased Env incorporation and viral replication. J. Biol. Chem. 278:33912-33919. - PubMed
    1. Bell, I., C. Ashman, J. Maughan, E. Hooker, F. Cook, and T. A. Reinhart. 1998. Association of simian immunodeficiency virus Nef with the T-cell receptor (TCR) zeta chain leads to TCR down-modulation. J. Gen. Virol. 79:2717-2727. - PubMed
    1. Benson, R. E., A. Sanfridson, J. S. Ottinger, C. Doyle, and B. R. Cullen. 1993. Downregulation of cell-surface CD4 expression by simian immunodeficiency virus nef prevents viral super infection. J. Exp. Med. 177:1561-1566. - PMC - PubMed
    1. Blagoveshchenskaya, A. D., L. Thomas, S. F. Feliciangeli, C. H. Hung, and G. Thomas. 2002. HIV-1 Nef downregulates MHC-I by a PACS-1- and PI3K-regulated ARF6 endocytic pathway. Cell 111:853-866. - PubMed

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