CEM15/APOBEC3G is a cellular protein required for resistance to infection by virion infectivity factor (Vif)-deficient human immunodeficiency virus (HIV). Here, using a murine leukemia virus (MLV)-based system, we provide evidence that CEM15/APOBEC3G is a DNA deaminase that is incorporated into virions during viral production and subsequently triggers massive deamination of deoxycytidine to deoxyuridine within the retroviral minus (first)-strand cDNA, thus providing a probable trigger for viral destruction. Furthermore, HIV Vif can protect MLV from this CEM15/APOBEC3G-dependent restriction. These findings imply that targeted DNA deamination is a major strategy of innate immunity to retroviruses and likely also contributes to the sequence variation observed in many viruses (including HIV).

The May 28, 2003 immediate early online version of this article (Cell 113, 803-809, 13 June 2003) contained one sentence that did not appear in the printed version. In the results subsection entitled "CEM15/APOBEC3G Is Incorporated into MLV Virions," a bracketed sentence appeared in the following context: The CEM15/APOBEC3G-mediated suppression of HIV infection is thought to be accomplished by protein transferred as a virion component from virus producing cells into target cells (curiously, such physical transfer of CEM15/ APOBEC3G appears uninhibitable by Vif) (Sheehy et al., 2002). The bracketed text was removed prior to publication of the definitive printed and corresponding online versions of the manuscript. It was our intention that this correction should have occurred in all versions of the article. The authors and Cell Press apologize for any inconvenience that may have been caused.

The antiretroviral activity of the cellular enzyme APOBEC3G has been attributed to the excessive deamination of cytidine (C) to uridine (U) in minus strand reverse transcripts, a process resulting in guanosine (G) to adenosine (A) hypermutation of plus strand DNAs. The HIV-1 Vif protein counteracts APOBEC3G by inducing proteasomal degradation and exclusion from virions through recruitment of a cullin5 ECS E3 ubiquitin ligase complex. APOBEC3G belongs to the APOBEC protein family, members of which possess consensus (H/C)-(A/V)-E-(X)24-30-P-C-(X)2-C cytidine deaminase motifs. Earlier analyses of APOBEC-1 have defined specific residues that are important for zinc coordination, proton transfer, and, therefore, catalysis within this motif. Because APOBEC3G contains two such motifs, we used site-directed mutagenesis of conserved residues to assess each region's contribution to anti-HIV-1 activity. Surprisingly, whereas either the N- or C-terminal domain could confer antiviral function in tissue culture-based infectivity assays, only an intact C-terminal motif was essential for DNA mutator activity. These findings reveal the nonequivalency of APOBEC3G's N- and C-terminal domains and imply that APOBEC3G-mediated DNA editing may not always be necessary for antiviral activity. Accordingly, we propose that APOBEC3G can achieve an anti-HIV-1 effect through an undescribed mechanism that is distinct from cytidine deamination.

The HIV-1 Nef protein is important for pathogenesis, enhances viral infectivity, and regulates the sorting of at least two cellular transmembrane proteins, CD4 and major histocompatibility complex (MHC) class I. Although several lines of evidence support the hypothesis that the Nef protein interacts directly with the cellular protein sorting machinery, the sorting signal in HIV-1 Nef has not been identified. By using a competition assay that functionally discriminates between dileucine-based and tyrosine-based sorting signals, we have categorized the motif through which Nef interacts with the sorting machinery as dileucine-based. Inspection of diverse Nef proteins from HIV-1, HIV-2, and simian immunodeficiency virus revealed a well-conserved sequence in the central region of the C-terminal, solventexposed loop of Nef (E͞DXXXL) that conforms to the consensus sequence of the dileucine-based sorting motifs found in cellular transmembrane proteins. This sequence in Nef NL4-3 , ENTSLL, functioned as an endocytosis signal when appended to the cytoplasmic tail of a heterologous protein.The leucine residues in this motif were required for the interaction of full-length Nef with the dileucine-based sorting pathway and were required for Nef-mediated down-regulation of CD4. These leucine residues were also required for optimal viral infectivity. These data indicate that a dileucine-based sorting signal in Nef is utilized to address the cellular sorting machinery. The data also suggest that an inf luence on the distribution of cellular transmembrane proteins may mechanistically unite two previously distinct properties of Nef: down-regulation of CD4 and enhancement of viral infectivity.

The maximal virulence of HIV-1 requires Nef, a virally encoded peripheral membrane protein. Nef binds to the adaptor protein (AP) complexes of coated vesicles, inducing an expansion of the endosomal compartment and altering the surface expression of cellular proteins including CD4 and class I major histocompatibility complex. Here, we show that Nef stabilizes the association of AP-1 and AP-3 with membranes. These complexes remained with Nef on juxtanuclear membranes despite the treatment of cells with brefeldin A, which induced the release of ADP-ribosylation factor 1 (ARF1) from these membranes to the cytosol. Nef also induced a persistent association of AP-1 and AP-3 with membranes despite the expression of dominant-negative ARF1 or the overexpression of an ARF1-GTPase activating protein. Mutational analysis indicated that the direct binding of Nef to the AP complexes is essential for this stabilization. The leucine residues of the EXXXLL motif found in Nef were required for binding to AP-1 and AP-3 in vitro and for the stabilization of these complexes on membranes in vivo, whereas the glutamic acid residue of this motif was required specifically for the binding and stabilization of AP-3. These data indicate that Nef mediates the persistent attachment of AP-1 and AP-3 to membranes by an ARF1-independent mechanism. The stabilization of these complexes on membranes may underlie the pleiotropic effects of Nef on protein trafficking within the endosomal system.

HIV-1 Nef interacts with cellular adaptor protein (AP) complexes and their medium (mu) subunits. However, the role of the dileucine-based sorting motif within Nef in these interactions has been incompletely characterized. Here, yeast two-hybrid assays indicated that HIV-1 Nef interacted not only with the mu subunits of AP-1 and AP-2, but also with that of AP-3. The interactions with mu1 and mu3 were markedly stronger than the interaction with mu2. Leucine residues of the sorting motif were required for the interactions with mu3 and mu2 and contributed to the interaction with mu1. Confocal immunofluorescence microscopy indicated that Nef, AP-1, and AP-3 (but not AP-2) were concentrated in a juxtanuclear region near the cell center, potentially facilitating interaction between Nef and the mu1 and mu3 subunits. However, leucine residues of the sorting motif were not required for this subcellular localization of Nef. These data suggest that the dileucine motif, required for optimal viral replication, functions through interactions with a variety of AP complexes, including AP-3, potentially by recruiting adaptor complexes to subcellular locations specified by additional determinants in the Nef protein.

Type 1 human immunodeficiency viruses encoding mutated nef reading frames are 10-to 30-fold less infectious than are isogenic viruses in which the nef gene is intact. This defect in infectivity causes nef-negative viruses to grow at an attenuated rate in vitro. To investigate the mechanism of Nef-mediated enhancement of viral growth rate and infectivity, a complementation analysis of nef mutant viruses was performed. To provide Nef in trans upon viral infection, a CEM derivative cell line (designated CLN) that expresses Nef under the control of the viral long terminal repeat was constructed. When nef-negative virus was grown in CLN cells, its growth rate was restored to wild-type levels. However, the output of nef-negative virus during the first 72 h after infection of CLN cells was not restored, suggesting that provision of Nef within the newly infected cell does not enhance the productivity of a nef-negative provirus. The genetically nef-negative virions produced by the CLN cells, however, were restored to wild-type levels of infectivity as measured in a syncytium formation assay in which CD4-expressing HeLa cells were targets. These trans-complemented, genetically nef-negative virions yielded wild-type levels of viral output following a single cycle of replication in primary CD4 T cells as well as in parental CEM cells. To define the determinants for producer cell modification of virions by Nef, the role of myristoylation was investigated. Virus that encodes a myristoylation-negative nef was as impaired in infectivity as was virus encoding a deleted nef gene. Because myristoylation is required for both membrane association of Nef and optimal viral infectivity, the possibility that Nef protein is included in the virion was investigated. Wild-type virions were purified by filtration and exclusion chromatography. A Western blot (immunoblot) of the eluate fractions revealed a correlation between peak Nef signal and peak levels of p24 antigen. Although virion-associated Nef was detected in part as the 27-kDa full-length protein, the majority of immunoreactive protein was detected as a 20-kDa isoform. nef-negative virus lacked both 27-and 20-kDa immunoreactive species. Production of wild-type virions in the presence of a specific inhibitor of the human immunodeficiency virus type 1 protease resulted in virions which contained only 27-kDa full-length Nef protein. These data indicate that Nef is a virion protein which is processed by the viral protease into a 20-kDa isoform within the virion particle.

The Nef protein from the human immunodeficiency virus (HIV) induces down-regulation of the CD4 and major histocompatibility complex class I molecules from the cell surface by interfering with the endocytic machinery. This work focuses on the interaction of HIV-1 Nef with the m1 chain of adaptor protein type 1 (AP1) complex and its contribution to the Nef-induced alterations of membrane trafficking. Two independent regions surrounding a disordered loop located in the C-terminal part of Nef are involved in m1 binding. Each region can separately interact with m1, and simultaneous point mutations within both regions are needed to abolish binding. We used CD8 chimeras in which the cytoplasmic tail was replaced by Nef mutants to show that these m1-binding sites contain determinants required to induce CD4 down-regulation and to target the chimera to the endocytic pathway by promoting AP1 complex recruitment. Ultrastructural analysis revealed that the CD8-Nef chimera provokes morphological alterations of the endosomal compartments and co-localizes with AP1 complexes. These data indicate that the recruitment by Nef of AP1 via binding to m1 participates in the connection of Nef with the endocytic pathway.