Reported Effects Of Hiv1 Nef On Immune Cell Function

The nef gene is found only in the primate lentiviruses HIV and SIV and codes for a protein of approximately 25-32 kDa and 206-265 amino acids. It is N-terminally myristoylated and generally found in the cytoplasm and membrane fractions of infected cells. Studies with rhesus monkeys infected with clones of SIV demonstrated that an intact nef gene was essential for maintenance of high virus loads and the development of disease.78 Additionally, investigators have described79 an epidemiological cohort of HIV-1-infected patients with nonprogressive disease in which nef sequences were not intact, which supports a role for Nef protein in disease progression. However, the potential pathogenic role of Nef may be dependent on the immune status of the host, since pathogenicity was still observed when Nef-attenuated SIV was used to infect neonatal macaques.80 A number of different biological activities have been described for Nef as well as a number of different mechanisms by which Nef might effect those activities. The majority of mechanisms described have focused on the interaction of Nef with intracellular regulatory proteins and have relied on either viral infection or nef gene transfection. The studies reviewed below and summarized in Table IV examined the potential extracellular role of Nef in immune dysregulation.

Early interest in an extracellular role for Nef was most likely restrained because of the known physical characteristics of the molecule and its common detection in infected cells in the cytoplasm or associated with cell membranes. However, antibodies to Nef have been routinely detected in HIV-infected patients beginning at the earliest stages of infection. Thus it is reasonable to conclude that the immune systems of patients are exposed to Nef proteins or peptide fragments early in infection; these might be shed either from virions or infected cells, expressed on the surface of infected cells, or released from dying cells.

Investigators had begun to look for antibodies to Nef and a potential

TABLE IV Potential Immunomodulating Activities Reported for HIV-1 Nef Protein

Activity References

In human PBMCs or lymphocytes

Inhibits proliferation in response to PHA 86

Induces Ig-secreting cells 93

Induces mRNA and protein secretion for IL-6 93

Induces V beta-specific expansion 96

Induces proliferation 94, 95, 97

Induces cytostasis or cytotoxicity in CD4+ cells 106, 107

Induces production of IL-2 and IFN-y 94

Inhibits production of IL-2 and IFN-y 108

Induces mRNA and protein for IL-10 105 In human bone marrow cultures

Inhibits proliferation of CFU-GM 110

correlation with disease as early as the late 1980s. Amiesen et al.,81 using purified recombinant Nef and Nef synthetic peptides, detected antibodies in the sera of HIV-1-seronegative, viral antigen-negative, and virus culture-negative individuals at risk for HIV infection. They concluded that antibodies to Nef could precede seroconversion. Reiss et al.82-83 examined antibody responses against Nef in a longitudinally studied cohort of 194 initially asymptomatic HIV-1-seropositive individuals and 72 individuals who sero-converted for antibodies to gag/env proteins. Antibodies to Nef were found in approximately 80% of the patients and antibodies persisted in about 85% of those patients. Although antibodies were detected early in infection, unlike the results of Amiesen et al.,81 they were rarely seen prior to antibodies to gag/env proteins. Although more cases of AIDS occurred in the Nef antibody-negative group (24%) than in the Nef-antibody-positive group (16%), the difference was not statistically significant. Wieland et al.84 examined the sera from 136 HIV-1-infected patients from different stages of disease. Although they found antibodies in a high percentage of patients, there was no obvious correlation with stage of disease or disease progression. In contrast, Rezza et al.85 found that patients who were persistently positive for antibody to Nef were statistically (p = .03) less likely to develop AIDS than those who were transiently or persistently negative.

Studies examining a potential role for extracellular Nef began to appear in the early 1990s. Fuji et al.86 reported studies utilizing Nef protein expressed in bacteria as a fusion protein with a phage gene product (Nef-gene 10). When PBMCs from healthy donors were incubated with IL-2 and Nef-gene 10, but not gene 10 alone, there was a decline in the CD4/CD8 ratio and in the response to PHA. The same result occurred with nylon wool-purified T cells. Although Nef-gene 10 inhibited the proliferation of CD4+ cells, it did not kill them and the inhibition of proliferation by Nef-gene 10 could be blocked by anti-Nef antibodies. These same investigators also reported87 that Nef was partly expressed on both acutely and persistently infected human T cell lines. They prepared monoclonal antibodies to recombinant Nef expressed in baculovirus as a truncated 22-kDa protein containing the middle to C-terminus of the native protein. The monoclonal antibodies reacted with both the 25- and 27-kDa forms of Nef and by flow cytometry reacted with the membranes of both fixed and unfixed human Molt4 T cells which had been infected with HIV-I. They suggested that the cell-surface form of Nef might play a role in depletion of CD4+ cells. In order to determine what portion of Nef might be expressed on the cell surface the investigators developed a series of monoclonal antibodies with identified reactivities to specific regions of the Nef molecule. Using these antibodies, they reported88 that intensity of cell surface staining on infected PBMCs was stronger with antibodies reactive to amino acid residues 158-206

and 192-206 than with antibody reactive with residues 148-157. They reported no staining with an antibody reactive to residues 1-33. Furthermore, they reported that binding of soluble Nef to the surface of uninfected CD4+ cells was blocked by antibodies to the C-terminal region (amino acids 158206 and 192-206) or by C-terminal synthetic peptides. Furthermore, they reported inhibition of syncytium formation between HIV-1-infected and uninfected cells by the same antibodies or peptides, suggesting that a cell surface domain of Nef could be important in pathogenic interactions between uninfected and infected cells.

It had been widely assumed that myristoylation of the N-terminus of Nef assured its association with membranes or insoluble fractions, although unmyristoylated Nef could be found in cell cytoplasm. Reports in 199489 and 199590 from two different laboratories offered an explanation as to how Nef fragments, perhaps biologically active, might find their way to the cell surface or the extracellular environment. Both laboratories reported that HIV-1 Nefwas cleaved into two fragments, of approximately 8 and 19 kDa, by the HIV-1 protease. The cleavage site was identified by both laboratories as being located between Trp57 and Leu58. Both recombinant Nef and Nef obtained from extracts of infected cells were subject to this specific cleavage. A subsequent study91 examined the physicochemical properties of Nef in virions and in infected cells. Quantitative analysis of radiolabeled HIV-1-infected cells and virions demonstrated that Nef was incorporated on the order of 10% of reverse transcriptase incorporation, which corresponded to 5-10 Nef molecules per virion. In infected cells Nefwas detected as a full-length, 27-kDa protein, while in virions, approximately 50% of the Nef was present as a 18-kDa protein. This virion-associated 18-kDa protein co-migrated with a 18-kDa protein generated by cleavage of purified Nef by HIV-1 protease. Cleavage of Nef in virion preparations was completely abolished by an HIV-1 proteinase inhibitor. These results have been confirmed92 by another laboratory, which found that the 18-kDa species of Nef was the major form in mature virions and that its generation was indeed dependent on HIV-1 protease. These studies also revealed that initial association of Nef with HIV-1 virions was dependent on its N-terminal myristoylation.

There have been several reports of Nef stimulating human peripheral blood cells. Chirmule et al.93 reported that recombinant Nefwas able to induce immunoglobulin-secreting cells in PBMC cultures ofHIV-1-seronegative donors. Pretreatment of Nef with a polyclonal anti-Nef antibody blocked its B cell stimulatory activity, as did monoclonal antibodies to LFA-1, ICAM-1, HLA-DR, and B7, suggesting that cell-to-cell contact was important in Nef-stimulated B cell activation. mRNA for IL6 was upregulated, primarily in monocytes, in Nef-treated PBMCs. Studies94-96 by another group have focused on the ability of Nef to function as a CD4 T cell superantigen. They reported that Nef-induced proliferation of human PBMCs was blocked by polyclonal antisera raised against Nef synthetic peptides and that responses were T cell-specific and required antigen presenting cells. Nef-stimulated cells produced the T helper 1 cytokines IL2 and IFN-y and activation did not require processing of Nef since paraformaldehyde-inactivated APC were still active. Their studies indicated that HIV could replicate in PBMCs activated by Nef and that this infection and replication could be blocked by polyclonal antisera to Nef. Using a simple amplification technique involving immobilized anti-VP antibodies, these investigators demonstrated expansion of VP T cells in cultures of Nef-treated PBMCs. They reported that expansion ofVp18 occurred in all of the donors tested, while VP5.3 expansion occurred in approximately 50% of the donors. The necessity for amplification suggested that Nef might be functioning as a weak, viralencoded superantigen.

The hypothesis that Nef might enhance infection and replication by stimulating T cell replication is indirectly supported by the studies of Novembre et al.97 In these studies in macaques, they used a variant of SIV, termed SIVsmmPBj, which induces an acute disease resulting in death in 514 days after infection. A molecular clone, PGj6.6 delta nef, was generated in which there was a deletion in the Nef-coding region. In general, PBj6.6 delta nef had markedly reduced replication abilities compared to the parental virus PBj6.6 when tested on pigtail or rhesus macaque PBMCs, although there was little difference in stimulated pigtail macaque PBMCs. Furthermore, PBj6.6 delta nef was unable to stimulate the in vitro proliferation of PBMCs, a feature characteristic for SIVsmmPBj.

In contrast to the earlier report by Torres et al.94 that Nef could induce the in vitro production of the cytokines IL2 and IFN-y, Beneviste et al.98 found that deletion of nef gene from SIV seemed to enhance the in vivo production of these same cytokines. They used semiquantitative RT-PCR to monitor cytokine expression in unmanipulated PBMCs during the acute phase of infection of cynomolgus macaques. In monkeys infected with the pathogenic SIVmac251, they found that IL2, IL-4, and IFN-y mRNAs were either weakly detectable or undetectable. In contrast, animals infected with a nef-truncated SIVmac251 exhibited overexpression of these cytokines. Both groups of monkeys exhibited similar rises in IL-10 mRNA expression coincident with the peak of viral replication. These results will be discussed further below.

In 1996 Collette et al.99 reported that the Nef protein of primate lenti-viruses shared sequence similarities with the immunosuppressive domain60,61 of the p15E TM of type C retroviruses of murine, feline, bovine, avian and human origin. This was an important observation because this immunosup pressive domain had been found to be highly conserved among virtually all of the retroviruses and had been postulated to play a role in the pathogenesis of these viruses. And yet, in the lentiviruses this region had only a limited degree of conservation in the gp41 TM. Studies with synthetic peptides corresponding to the immunosuppressive domain (CKS17) of p15E had suggested a potential role for cytokine dysregulation as a mechanism for its biological effects (reviewed in ref. 100). Among the in vitro activities identified for CKS-17 on human cells are inhibition of induction of IL2, IL-1, IFN-y and TNF-a and enhancement of the induction of IL-10 The potential of the CKS-17 immunosuppressive domain to effect a Th1 ^ Th2 type of cytokine shift, coupled with the now-identified similarity ofa portion ofNefto CKS-17, was provocative since several investigators had already suggested that such a shift might be a critical step in HIVpathogenesis.101-103 Furthermore, Clerici et al.104 had reported that IL-10-specific mRNA was upregulated (although marginally) and increased levels of IL-10 were produced from PBMCs of HIV-infected individuals compared with uninfected individuals. In those studies they also reported that those patients whose T helper cell function was more severely suppressed produced higher levels of IL-10 and that defective in vitro antigen-specific T helper functions could be reversed by addition of anti-IL-10 antibodies. Thus, the recently identified structural similarities between Nef and the CKS-17 region of p15E, strong evidence suggesting a role for Nef in the pathogenesis of HIV-1 infections, and evidence suggesting a Th1 ^ Th2 type of cytokine shift potentially effected by IL-10 have led to studies investigating the potential effects of extracellular Nef on cytokine regulation and lymphocyte proliferation.

Brigino et al.105 recently studied the ability of recombinant Nef to effect cytokine induction in human monocytic THP-1 cells or freshly isolated PBMCs. In those studies they found that extracellular Nef produced in either yeast (Saccharomyces cermisiae) orbacteria (Escherichia coli) induced, in adose-dependent manner, the release of IL-10 into the supernatants of Nef-treated human PBMCs. They could detect IL-10 with as little as 10 ng/ml (370 pM) recombinant Nef and although the E. coli-derived material also induced IL-10 it was not quite as effective as the yeast-produced material (E. Brigino, personal communication), which might be a function of the N-terminal myris-toylation on the yeast protein or the ability ofyeastderived material to retain a more natural conformation. In addition to protein expression, Brigino et al.105 also demonstrated by RT-PCR the induction of mRNA for IL-10 but not for IL2, IL-4, IL-5, IL-12 (p40), IL-13, or IFN-y in PBMCs treated for 3 h with E. coli-derived Nef. By Northern blot analysis they estimated a threefold increase in mRNA for IL-10 at 24 h after Nef stimulation.

A report the previous year by Fujii et al.105 was significant in that they reported, apparently for the first time, the detection of soluble Nef in the sera of HIV-1-infected individuals. Using an antigen capture ELISA they detected soluble Nef in the sera of 27 of 32 HIV-infected individuals, but not in any of the 28 healthy individuals used as controls. The five sera in which Nefwas not detected contained high titers (>1:5000) of anti-Nef antibodies, whereas the other sera contained no levels of anti-Nef antibodies. Importantly, in the sera of 21 of 2'7 individuals positive for Nef the concentrations were 5-10 ng/ml, which was close to the concentration at which Brigino et al.105 were able to stimulate IL-10 release from normal human PBMCs. This concentration is also similar to that with which we have observed (Fig. 1) significant inhibition of anti-CD3-stimulated proliferation of normal human PBMCs with recombinant (E. coli) Nef. In those studies we found that recombinant Nefinhibits in a dosedependent manner the proliferation of normal PBMCs in response to anti-CD3 monoclonal (OKT3; 50 ng/ml) antibody. Although Fujii etal.106 and

FIGURE 1. Effect ofrecombinant HIV-1 Nef on proliferation ofhuman peripheral blood mono. nuclear cells in response to OKT3 stimulation. Recombinant Nefwas expressed in E. coli, purified to homogeneity, and contaminating endotoxin removed by multiple passages over DeToxi-Gel (Pierce, Rockford, IL) until endotoxin levels were ng/ml. Human PBMCs were isolated and cultured for 72 h at 2 x 105 total cells/well in 200 ^l in 96-well, flat-bottom tissue culture plates as previously described.6162 Recombinant Nef was added at the indicated concentrations at the beginning of the cultures and OKT3 monoclonal antibody was added to each well at a final concentration of 50 ng/ml. One-half ^Ci of 3H-thymidine (6.7 Ci/mmol; Amersham) was added to each well for the last 6 h of culture and the amount ofincorporated radioactivity determined by harvesting the cells onto glass fiber filters and counting the filters in a liquid scintillation spectrophotometer. Results represent the means and standard errors of quadruplicate samples. Similar results were observed in at least two other experiments.

FIGURE 1. Effect ofrecombinant HIV-1 Nef on proliferation ofhuman peripheral blood mono. nuclear cells in response to OKT3 stimulation. Recombinant Nefwas expressed in E. coli, purified to homogeneity, and contaminating endotoxin removed by multiple passages over DeToxi-Gel (Pierce, Rockford, IL) until endotoxin levels were ng/ml. Human PBMCs were isolated and cultured for 72 h at 2 x 105 total cells/well in 200 ^l in 96-well, flat-bottom tissue culture plates as previously described.6162 Recombinant Nef was added at the indicated concentrations at the beginning of the cultures and OKT3 monoclonal antibody was added to each well at a final concentration of 50 ng/ml. One-half ^Ci of 3H-thymidine (6.7 Ci/mmol; Amersham) was added to each well for the last 6 h of culture and the amount ofincorporated radioactivity determined by harvesting the cells onto glass fiber filters and counting the filters in a liquid scintillation spectrophotometer. Results represent the means and standard errors of quadruplicate samples. Similar results were observed in at least two other experiments.

Okada et al.107 have reported that soluble Nefis cytotoxic for CD4+ cells from PBMC, they found that cytotoxicity required Nef cross-linking, which they effected with anti-Nefantibody. We have not observed cytotoxicity in our Nef-treated PBMCs, as determined by trypan blue dye exclusion, a result sup ported by our observation (data not shown) that soluble Nef had little or no inhibition on mitogen-stimulated (PHA, ConA) proliferation of PBMCs under the same conditions.

We have begun to examine the potential role of the CKS-17-related region of Nef in its potential immunosuppressive effects on human lymphoid cells. A peptide (MN10042) was synthesized to correspond to the region (amino acids 79-104) of HIV-1 Nef which was partially homologous to the immunosuppressive CKS-17 region of p15E TM of type C retroviruses. Although initial studies61 with CKS-17 had indicated that biological activity required coupling to a protein carrier, subsequent studies revealed that dimerization of peptide monomers through a naturally occurring C-terminal cysteine was sufficient to generate biologically active molecules. For our studies examining the potential active site of Nef, we prepared peptide monomers (corresponding to HIV-1 Nef amino acid residues 79-104) with a C-terminal cysteine (not naturally occurring) and then either dimerized them (MN10042) or, as a control, prepared monomer in which the cysteine was blocked (MN10041.5). As shown in Fig. 2, MN10042 inhibited in a dose-dependent manner the tetanus toxoid-stimulated proliferation of human PBMCs, whereas the control peptide, MN10041.5, had no activity. This effect was not simply a result of the presence of an active disulfide, since we previously found (data not shown) that other peptide dimers with similar amino acid compositions had no effect on PBMC proliferative responses. Although the concentration of MN10042 required for 50% inhibition was approximately three orders of magnitude greater than that required with soluble recombinant Nef, that result was not unexpected. Our experience has been that peptides are generally much less active than the native proteins from which they are derived, probably because their conformations are not stably fixed in solution.

More recently, Haraguchi etal.108 have shown thatMN10042 inhibits the production of IL-2 and IFN-y from human PBMCs stimulated with either OKT3 or staphylococcal enterotoxin A, but not from PBMCs stimulated with phorbol-12-myristate 13-acetate plus ionomycin. Expression of mRNAs for both cytokines was also inhibited in the presence of MN10042. This result would seem to be in agreement with the results reported by Beneviste et al.,98 who described increased production of IL2 and IFN-y in macaques infected with Nef-deleted SIV compared to the wild-type virus. Although MN10042 suppresses IL2 and IFN-y as had been observed for CKS-17, there are mechanistic differences since CKS-17 has been shown to induce dramatic increases

40000

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FIGURE 2. Effects of HIV-1 Nef-derived peptides on proliferation of human peripheral blood mononuclear cells in response to tetanus toxoid. Monomeric peptide corresponding to amino acid residues 79-104 of HIV-1 Nef was synthesized with a C-terminal cysteine (MTYKAAVDLSHFLKEKGGLECLIHSQ-C) on aRAININ P3 peptide synthesizer andpurified to >95% by reversed-phase HPLC. Homogeneity was confirmed by amino acid analysis and mass spectroscopy. Peptide dimers (MN10042) or peptides with a blocked C-terminal cysteine (MN10041.5) were prepared as previously described.109 Purity and homogeneity of all peptides were confirmed by mass spectroscopy. Human PBMCs were isolated and cultured as described for Fig. 1 except that the cultures were incubated for 144 h. MN10042 (solid bars) or MN10041.5 (open bars) was added at the indicated concentrations at the beginning of the cultures. Tetanus toxoid was added to each well to a final concentration of 4 LF/ml. Thymidine incorporation was determined as for Fig. 1. Results represent the means and standard errors of quadruplicate samples. Similar results were observed in at least two other experiments.

FIGURE 2. Effects of HIV-1 Nef-derived peptides on proliferation of human peripheral blood mononuclear cells in response to tetanus toxoid. Monomeric peptide corresponding to amino acid residues 79-104 of HIV-1 Nef was synthesized with a C-terminal cysteine (MTYKAAVDLSHFLKEKGGLECLIHSQ-C) on aRAININ P3 peptide synthesizer andpurified to >95% by reversed-phase HPLC. Homogeneity was confirmed by amino acid analysis and mass spectroscopy. Peptide dimers (MN10042) or peptides with a blocked C-terminal cysteine (MN10041.5) were prepared as previously described.109 Purity and homogeneity of all peptides were confirmed by mass spectroscopy. Human PBMCs were isolated and cultured as described for Fig. 1 except that the cultures were incubated for 144 h. MN10042 (solid bars) or MN10041.5 (open bars) was added at the indicated concentrations at the beginning of the cultures. Tetanus toxoid was added to each well to a final concentration of 4 LF/ml. Thymidine incorporation was determined as for Fig. 1. Results represent the means and standard errors of quadruplicate samples. Similar results were observed in at least two other experiments.

in intracellular cAMP, while MN10042 appears to act through a cAMP-independent mechanism.108109

Still another mechanism which has been proposed for Nef-associated immunosuppression is the effect of Nef on hematopoiesis. Calenda et al.110 reported that supernatants from HIV-1 non-productivity infected cultures inhibited CFU-GM in clonogenic assays and that the active, growth-inhibitory factor was Nef. Anti-Nef antibodies were able to block the activity in the culture supernatants and recombinant Nef was able to mimic the activity from the culture supernatants. Furthermore, the authors confirmed the involvement of Nef by demonstrating that culture supernatants generated with Nef-deficient virus were inactive.

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