A Genetic Search for Cellular Factors Involved
in CD4 Down-regulation by HIV-1 Nef

Yibin Kang

Department of Genetics &
Cell and Molecular Biology Program

Duke University Medical Center

April 28, 1997

Abstract
Although Nef protein encoded by primate immunodeficiency viruses plays a critical role in the viral pathogenesis in vivo, the molecular basis of its function remains to be discovered. Cell surface CD4 down-regulation in cell culture is the most readily reproducible and least controversial property of Nef and this process is mediated by endocytosis and degradation of CD4 in lysosome. However, the detailed mechanism of this Nef-dependent cell surface CD4 down-regulation pathway is still poorly understood. Using a random antisense homologous knockout strategy, a genetic screening is proposed here to identify cellular factors involved in the down-regulation of CD4 by HIV-1 Nef. The identification of these genes and detailed analysis of their functions will provide new insights into the molecular mechanism of HIV-1 pathogenesis.

Background and significance:
The genome of lentiviruses are characterized by the presence of several accessory genes in addition to the prototypic retroviral gag, pol, and env sequences (Fig 1A; for review see 1). Among those, nef open reading frame is found only in primate lentiviruses (2, 3). Nef is dispensable for virus growth in cultured cells, but studies of rhesus monkeys infected with nef-mutated strains of simian immunodeficiency virus (SIV) have demonstrated that there is a strong selective pressure on the virus to maintain a function nef open reading frame (Figure 1B, ref 4). In several long-term HIV-infected non progressors, the efficient control of HIV-1 infection is associated with deletions in nef (5). Therefore, nef is believed to play a critical role in maintaining high viral load and AIDS progression. HIV-1 Nef is a 206 amino acid long cytoplasmic protein that associated with the plasma membrane through an N-terminal myristic acid (6). Nef is expressed early in the HIV-1 replication cycle and is the predominant viral early gene product in terms of its level of expression (7). However, the role of Nef in the viral life cycle remained ill-defined. While data from several groups have demonstrated that Nef can enhance the infectivity of HIV-1 virions released by some Nef expressing cells (8, 9), Nef has been found to be dispensable for efficient viral replication in other culture settings (10, 11). It has been consistently observed that Nef down-regulates cell surface level of CD4, the primary viral receptor by as much as one magnitude (6, 12, 13; Fig 1C, Fig 2). This effect is reproducible in a variety of lymphoid and nonlymphoid cells and is known to occur posttranscriptionally, because levels of CD4 mRNA and steady level of CD4 protein are unaltered in the presence of Nef (12). This Nef activity, which renders cells resistant to viral superinfection (13), has the potential to increase virus replication by facilitating release of progeny virions (14). Nevertheless controversy still exists as to whether CD4 down regulation plays an important role in vivo among many proposed functions of Nef, or instead is only a minor secondary effect of Nef. Mutational analysis of Nef has demonstrated that infectivity enhancement and CD4 down regulation can be functional segregated (15, 16), thus strongly suggesting that Nef has at lease two distinct biological activities, both of which may be important. In addition, several groups has presented data arguing that Nef can affect the activation state of infected cells and signaling pathways in helper T cells (17, 18, 19). It remains unclear whether this represents a separate, third biological phenotype or whether the effects of Nef on cell activation are instead correlated with effects on virion infectivity and/or CD4 internalization, even though at least one report has point out the ability of Nef to disrupt CD3 signaling is independent of its function in CD4 down regulation (20). Importantly, among these reported functions of Nef, CD4 down-regulation is the most readily reproducible and least controversial property of Nef in cell culture.
The CD4 glycoprotein is crucial for antigen-driven helper T cell signaling and is the primary receptor for HIV-1 and SIV on both T-cells and macrophage. The hypothesis that Nef could down-regulate cell-surface expression of the CD4 receptor was first proposed by Guy et al. (12) and confirmed subsequently by others (21, 22). This apparent Nef-induced internalization of CD4 was, however, independent of three intracytoplasmic CD4 serine residues known to be involved in phorbol ester mediated down-regulation of CD4 through endocytosis and degradation of CD4 in lysosome (23), thus suggesting a novel mechanism of action. The necessary and sufficient CD4 target for Nef internalization is distinct from the sequences within CD4 that are known to interact with the two factors, i.e. Lck, a protein tyrosine kinases and PKC, which are known to affect the cell surface expression of CD4 (24). Nef mediated down-regulation of CD4 can occur in both non lymphoid and non-human cell lines (25), implying a highly conserved pathway of internalization. On the other hand, Nef-induced internalization of CD4 is a highly specific consequence of Nef expression. Specificity is conferred by sequences located in a 38 amino acid intracytoplasmic domain of CD4, including a dileucine motif that is critical for Nef-medicated endocytosis (26, 27). With the possible exception of MHC I, whose responsiveness to Nef has been controversial (28), none of the many other cell surface molecules that have been examined so far was shown to be internalized in response to Nef expression (29, 13). It is therefore apparent that CD4 down-regulation must involve the sequence specific recognition of the CD4 intracytoplasmic domain by Nef, by a cellular factor activated by Nef, or by combination of both. Although the Nef protein are know to induce the efficient internalization and degradation of cell surface CD4, it remains unclear whether this involves a direct interaction between Nef and CD4. The demonstration that different Nef protein recognized distinct target sites within the intracellular domain of CD4 suggest that it's unlikely that the CD4 intracytoplasmic domain is recognized exclusively by a conserved cellular Nef cofactor(s) (30). However direct binding of CD4 by Nef has been difficult to demonstrate, although evidence supporting such an interaction has been shown indirectly by using yeast two hybrid system (31), using overexpression in insect cells(32), and using NMR spectroscopy (33). While these studies do provide evidence for the specificity of the observed CD4:Nef interaction, the observed in vitro binding displayed an affinity (Kd ~1mM) that is significantly lower than is normally seen for physiologically relevant interactions (33). Therefore it seems more likely that a cellular cofactor(s) is involved in the recognition between CD4 and Nef. Identification of a cellular cofactor that specifically facilitate CD4-Nef interaction will not only open a new window to study the molecular mechanism of CD4 down-regulation by Nef, but also provide a strong argument that CD4 down-regulation is an important primary function of Nef instead of being a minor secondary effect.
The detailed pathway of CD4 internalization to lysosome in the process of down-regulation by Nef remains undefined. The observation that most cell surface is not the target of Nef-mediated down regulation suggest that this pathway has some unique features compared to other surface protein internalization processes. Either or not the Nef-dependent pathway finally converge with other pathway, it will be interesting to define the detailed steps or Nef mediated endocytosis and degradation of CD4. Therapeutic reagents that specifically blocked this Nef-mediated pathway will provide a new alternative of anti-HIV drugs and increase the chance of winning the war against HIV.
The specific aim of this proposed study, therefore, is to identify cellular factors whose mutation disrupt the down-regulation of CD4 by Nef. In particular, the cellular cofactor(s) that mediates CD4:Nef interaction and Nef-specific lysosome targeting are of major interests in this proposed study.

Experimental design:
An apparent strategy to identify cellular factors involved in CD4 down regulation by Nef is selecting mutants that show normal surface CD4 level even when Nef is constitutively expressed in the cell. However, the diploid genome of human cells complicates the mutagenesis and selection of this recessive phenotype. Recently a novel strategy was established that used regulated antisense RNA initiated within a retrovirus-based gene search vector to identify previously unknown mammalian autosomal genes whose homozygous inactivation are associated with a defined recessive phenotype (34). Application of this strategy has successfully lead to the discovery of a novel tumor suppressor gene tsg101 that has surprising importance in the tumorigenesis of human breast cancer (34, 35). A modified strategy is proposed here to identify genes whose homozygous disruption inactivate the Nef mediated CD4 down-regulation pathway and restore the normal surface CD4 level.
The construction of a retroviral gene search vector (pGSV) and a gene search cell line (GS) is shown in figure 3 and figure 4 respectively. The GS cell line is derived from human CEM T cell line, which constitutively expresses cell surface protein CD4 and IL-2 receptor a chain. A Tet-off promoter controlled Nef expression construct was integrated into genomic DNA and the Nef expression from this construct can be finely tuned by the adjusting the concentration of doxycycline in the medium. Regulatory proteins of the ecdysone-inducible promoter system, which is responsive to the muristerone A level in the medium, are also constitutively expressed in this cell line. Both Tet-Off and ecdysone inducible expression systems are highly sensitive system to regulate gene expression in mammalian system. These system feature low basal expression/high inducibility and are inert to mammalian physiology and do not have any pleiotropic effects. The Tet-Off and ecdysone inducible expression system are commercially available from Clontech Inc. and Invitrogene Inc. respectively. This cell line will be tested to confirm the integrity of the regulatory system using transient transfection assay.
pGSV, a Moloney murine leukemia virus-derived retroviral gene search vector containing the b-geo (36) reporter gene was shown in Figure 4A. This vector will be introduced into GS cell line, where it will integrate at multiple chromosomal sites. As the proviral state of the retrovirus contained in pGSV lacks an enhancer and promoter in its long terminal repeats (LTR) (37), expression of b-geo in cells containing the provirus is dependent on transcription directed by the adjacent chromosomal promoter (Figure 4B). An adenovirus-derived splice acceptor site located 5' to b-geo fuses b-geo mRNA to exons of chromosomally encoded transcripts. A splicing event that occurs between chromosomal splice donor site and the splice acceptor site located at the 5' end of b-geo coding sequences will create a chimeric transcript of chromosomal exons and b-geo (Figure 2B). Such expression will yield resistance to the antibiotic G418 and production of b-galactosidase. The ecdysone-inducible promoter are 5' to the splice acceptor site and in reverse (antisense) orientation to b-geo; transcription from this promoter can be activated in trans by muristerone A. The system is designed so that antisense RNA from the regulated SV40 promoter will inactivate b-geo fusion transcripts initiated in chromosomal genes that contain the pGSV-derived provirus and concomitantly will inactivate transcripts from other copies of these chromosomal genes. Clones in which such homologous gene inactivation leads to an identifiable phenotype (normal CD4 level in the presence of Nef) can be isolated form a heterogeneous cell population.
What mutations can we expect that can result in normal CD4 level even when Nef is expressed in the cell? Some possible mutants with such a phenotype are summarized in figure 5A. Group A1 has mutations in cofactor(s) that mediates Nef:CD4 interaction in the cytoplasmic membrane and group A2 has defects in the Nef-specific portion of the endocytosis and lysosomal targeting pathway. The purpose of this proposal is to identify groups A1 and A2 mutants specifically without selecting other groups of mutants at the same time, even though these other mutations may be interesting targets for further research. Group B, C and D mutants does not involved in Nef-mediated CD4 down-regulation pathway. Instead they are mutants that increase cell surface CD4 level and compensate for the lose of CD4 caused by Nef down regulation. Therefore these groups of mutants can be easily distinguished from other mutants by virtue of their abnormally high level of CD4 when Nef expression is turned off (Figure 5B). Group E mutants has defects in the possible portion of the endocytosis/degradation pathway that's common to many other cell surface proteins such as IL-2 receptor a chain. This group of mutants exhibit higher IL-2Ra level than wild type cells, which can serves as a unique property to sort out this group of mutants. Group F mutants has lower level of functional Nef protein properly targeted onto cytoplasmic membrane. Since Nef protein is expressed from a introduced expression construct that has no intron, the mutations on the Nef gene itself is unlikely to occur. Other mutations such as those who disrupt the myristyolation and targeting of Nef to the cytoplasmic membrane can be distinguished from group A1 and A2 mutant by their low level of membrane associated Nef protein.
Figure 6 shows the procedures that will be carried out to identify clones in which random homologous knockout of a cellular gene results in mutants whose CD4 level are irresponsive to Nef. GS cells will be infected with retrovirus particles that are derived from pGSV and produced by helper cells that harbor genes necessary for viral production and packaging. Cell expressing b-geo, which contain the provirus integrated at a transcriptionally active chromosomal site, will be selected from G418 resistance or collected by florescence-activated cell sorter (FACS) for production of b-galactosidase. To minimize false positive and increase the selection efficiency and specificity, two sequential FACSes will be carried out to select a starting population of cells from b-geo expressing cells for subsequent screening. The first FACS selects for cells with normal level of IL-2Ra when cells are grown in muristerone containing medium, therefore eliminates group E mutants mentioned above. Since retroviral integration disrupt one copy of the targeted gene even when antisense RNA is not synthesized, some retroviral insertions in genes whose function is dosage-dependent will cause a moderate increase of CD4 even though the other copy of the gene is still functional. Therefore the second FACS will be done to select those cells with completely or moderately down regulated CD4 in normal medium. This second FACS will eliminate cells that constitutively express high level of CD4 in the presence of Nef.
Repeated rounds of three-set FACS selections will then be applied to this population of cells (see Figure 6 for details). Selection will be repeated until the enriched population of cells show clear expected surface CD4 profile as those of group A1/A2 mutants shown in Figure 5B. This final population of selected mutants will then be plated to form colonies.
Initial analysis of each colonies will includes FACS analysis of surface CD4 and IL-2Ra level when the antisense RNA are turn on or off, Northern blot and Southern blot to confirm integration of retroviral vector into single genomic site and production of a single fusion transcript. The acid-elution assay (38) will provide further proof that homologous knockout of these genes indeed affect the endocytosis of CD4 by Nef, while the CD4 endocytosis induced by phobol ester is not affected. The expression level of membrane associated Nef level will also be assessed to eliminate group F mutants. Gene cloning will be subsequently carried out on those best candidate colonies by RT-PCR and screening of cDNA library with sequence derived from RT-PCR product as the probe.
With the sequence of candidate gene at hand, further characterization and functional study of these genes will follow. Some group A1 candidate gene product may show co-localization with CD4 and/or Nef in cytoplasmic membrane. In addition genes will homology to cytoskeleton proteins are likely candidate as the mediator of Nef:CD4 interaction or the internalization. For group A2 genes their role in Nef mediated CD4 down regulation will require more effort to elucidate. In general, the importance of these genes in CD4 down-regulation can be confirmed by transient transfection experiments in which high level expression of these gene increase turnover rate of CD4, while expression of antisense RNA of these genes will block the down-regulation of CD4 by Nef. Some dominant negative mutants that can still recognize CD4 or Nef, but are no longer functional in inducing the endocytosis will serve as useful tools in dissecting the multiple biological phenotype of Nef.

Discussion
Traditionally the genetic method of identifying mammalian genes associated with a defined phenotype in cell culture system is mainly depend on "gain of function" analysis, i.e. introduction of a gene into a system produce a new phenotype that previously was not observed in that cell line. Such a method has the advantage of circumventing the difficulty of homologous mutation in a diploid genome and indeed has produced many successful examples such as the discovery of HIV entry cofactor Fusin (39). However this approach has several apparent disadvantages. Sometimes it's impossible or difficult to obtain a negative-phenotype system to start with. In addition, it is difficult to analyze phenotypes that require synergistic effect of several factors simultaneously. With the combination of antisense knockout technique and efficient recombinant retroviral gene delivery system, the "lose of function" strategy will provide a valuable alternative for genetic analysis of cellular function in mammalian system.
Retroviral vectors are becoming a powerful weapon to attack some difficult questions in the molecular biology of retroviruses (see Figure 7B for another application of this strategy in searching for HIV-1 Tat cofactor). Still, many improvement can be made to make these vector more efficient and versatile. One of the major concerns about the strategy described in this proposal is the efficiency of "productive" retroviral integration that can produce fusion transcripts. Whereas retrovirus integration are largely random events, preference for integration at the 5' ends of functional genes has been observed (40). Use of the b-geo reporter gene allows cells containing integrations in transcriptionally active genes to be selected and also allows monitoring of the effects of antisense RNA on transcripts initiated in the flanking chromosomal DNA sequence. Reversibility of antisense inhibition and the CD4 level phenotype was accomplished in these experiments by ecdysone-inducible promoter. However, there are still some drawbacks about the application of this retroviral vector. First of all, this method can not identify genes without intron, even though such genes are rare in mammalian genome. In additions, small genes and genes that are unfavorable for retroviral integration will be difficult to identify too. A new retroviral vector based on transcription of cDNA library cloned in a antisense orientation, as shown in figure 7A, can be a valuable addition to the arsenal of gene search retroviral vectors. Such a vector can be produced easily with only few modifications of a highly efficient cDNA-based retroviral vector that has been available recently (41). This antisense cDNA-based vector, even though has the disadvantage in detecting gene with low expression level and in monitoring of antisense knockout effectiveness, can compensate the drawbacks of the fusion transcript-based vector.
Homologous knockout of some cellular gene that involves in Nef-mediated CD4 down-regulation may produce a vital effect on the cell and make such mutants impossible to isolated. Whereas leaky expression of these genes by tuning down the strength of ecdysone-inducible promoter can increase the chance of identifying viable mutants, it will also make the selection less sensitive. An alternative "gain of function" strategy can be applied here: expression of Nef in the GS cell line can be titrated to define the threshold level of Nef from which further increase of Nef expression will no longer results in subsequent further decrease of cell surface CD4. In such a Nef saturated cell, the cellular proteins involved in the down-regulation process will become limiting factors and introduction of a cDNA expression construct will produce a "super down-regulation" phenotype.

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