Nef does not contribute to replication differences between R5 pre-AIDS and AIDS HIV-1 clones from patient ACH142
© Olivieri et al; licensee BioMed Central Ltd. 2008
Received: 13 May 2008
Accepted: 29 May 2008
Published: 29 May 2008
AIDS-associated, CCR5-tropic (R5) HIV-1 clones, isolated from a patient that never developed CXCR4-tropic HIV-1, replicate to a greater extent and cause greater cytopathic effects than R5 HIV-1 clones isolated before the onset of AIDS. Previously, we showed that HIV-1 Env substantially contributed to the enhanced replication of an AIDS clone. In order to determine if Nef makes a similar contribution, we cloned and phenotypically analyzed nef genes from a series of patient ACH142 derived R5 HIV-1 clones. The AIDS-associated Nef contains a series of residues found in Nef proteins from progressors . In contrast to other reports [1–3], this AIDS-associated Nef downmodulated MHC-I to a greater extent and CD4 less than pre-AIDS Nef proteins. Additionally, all Nef proteins enhanced infectivity similarly in a single round of replication. Combined with our previous study, these data show that evolution of the HIV-1 env gene, but not the nef gene, within patient ACH142 significantly contributed to the enhanced replication and cytopathic effects of the AIDS-associated R5 HIV-1 clone.
The nef gene of HIV-1 plays a pivotal role in the pathogenesis of AIDS [4–8]. For example, patients infected with nef-deleted HIV-1 exhibited much slower progression to AIDS [6, 9–11]. The nef gene is important for viral replication in mature T cells [12–16] and macrophages [14, 17–19]. When thymocytes are infected, Nef plays a role in increasing the cytopathic nature of the virus [20–24]. The importance of Nef is further corroborated by observations of immune dysfunction in nef-transgenic mice [25–28].
Several functions have been assigned to Nef although the role of each in disease progression has not been firmly established (for reviews see: [21, 29–37]). We chose to focus on Nef's abilities to downmodulate CD4  and cell surface MHC-I A and B molecules [39, 40], and its ability to enhance viral infectivity [12, 41, 42]. These functions have been well studied by several labs and in various cell types and systems [17, 43–50]. Nef mediated enhancement of infectivity may be due to Nef downmodulation of cell surface CD4, allowing more efficient Env incorporation into HIV-1 particles [51, 52]. Enhancement of infectivity may also occur when Nef is present in CD4 negative producer cells [12, 53–56]. In this case, enhancement appears to act at a post-entry, pre-integration step in the viral life cycle [57, 58] and may be related to interaction of the viral pre-integration complex with the actin cytoskeleton . Downmodulation of MHC-I A and B molecules protects cells from lysis by HIV-1 specific cytotoxic T cells . The ability to avoid the immune system may be important in establishment and maintenance of infection.
Kirchhoff and colleagues compared the predicted amino acid sequences of Nef proteins from progressors with those of non-progressors and found that certain residues characterize Nef sequences from each type of patient . When compared to non-progressor Nefs, progressor Nefs were better able to downmodulate CD4 and less able to downmodulate MHC-I molecules, and also may have an increased ability to enhance HIV-1 infectivity [1, 2].
Previously, we demonstrated that the ACH142 AIDS clone *E11 was better able to replicate and cause cytopathic effects in human fetal thymus-liver grafts implanted in severe combined immune deficient mice (SCID-hu thy/liv mice), than the pre-AIDS clones, 8G9 and 32D2 . In an analysis parallel to this study, we examined the phenotypes of the env genes from these clones and determined that the AIDS associated env likely contributed to the observed replication differences between the AIDS clone and the pre-AIDS clones . In order to determine if nef made a similar contribution, ACH142 nef genes were amplified from PHA-activated PBMC infected with the HIV-1 clones ACH142-*E11, 32D2, and 8G9. The one kilo base nef/LTR products were gel purified and inserted into the pGEM-T vector (Promega). Six *E11, six 32D2 and three 8G9 full-length nef genes were sequenced.
To elucidate the significance of these differences and to assign phenotypes to each Nef protein, we inserted each consensus nef gene into an actin promoter driven expression vector (pA-nef). Next we analyzed the ability of each Nef to downmodulate cell surface CD4 and MHC-I A2 molecules on the T lymphoblastoid cell line, SupT1. Electroporation and flow cytometric analysis of SupT1 cells bearing pA-nef expression vectors was done as previously described . Two μg of pCMV-EGFP and 10 μg of each pA-nef expression plasmid, or empty pA vector were introduced into SupT1 cells by electroporation. The cells were then plated in 10 cm dishes and cultured for 24 hours. Subsequently, the cells were incubated with CD4-PerCP and MA2.1-PE monoclonal antibodies to detect CD4 and the A2 allele of MHC-I by flow cytometry. Data were collected with a FACSCalibur instrument and the GFP+ population was analyzed for CD4 and MHC-I surface expression with CellQuest software.
Our results suggest that the AIDS associated nef gene studied here does not significantly contribute to the enhanced replication and cytopathic effects of the AIDS associated *E11 R5 HIV-1 clone for the following three reasons. It is highly conserved at almost all known sites within the Nef sequence that are implicated in functional interactions. It does not downmodulate CD4 to a greater extent than pre-AIDS Nefs, nor does it more greatly enhance infectivity in a single round assay compared to the pre-AIDS Nefs from the same patient. Combined with our previous study, we conclude that Env, but not Nef contributes to the enhanced replication of the R5, AIDS-associated HIV-1 clone ACH142-*E11 compared to two pre-AIDS R5 HIV-1 clones derived from the same patient .
Previous studies indicated that Nef proteins with high progression scores had enhanced ability to downmodulate CD4, reduced ability to downmodulate MHC-I and increased ability to enhance HIV-1 replication compared to Nef proteins with lower progression scores [1–3]. The Nef proteins of patient ACH142 derived R5 HIV-1 clones displayed a chronological increase in Nef progression score as predicted, but the phenotype of these genes differed from the predicted phenotypes described above. The AIDS associated Nef protein studied here had a higher progression score (+5) than the pre-AIDS Nef proteins derived from the same patient (+2 and +3), but did not show an increased ability to downmodulate CD4 or to enhance infectivity. Moreover, the AIDS associated Nef protein had greater ability to downmodulate MHC-I A2 molecules. This is likely not explained by differences in Nef expression because the *E11 Nef downmodulated MHC-I to a greater extent despite being expressed at a slightly lower level. One explanation for the discrepancy between our results and those previously reported by others may be that nef genes from CXCR4-tropic (X4) HIV-1 isolates have the phenotypes previously reported [2, 3] but nefs from R5 HIV-1 clones show the phenotypes demonstrated here. Most of the progressor nefs used in previous studies were likely derived from X4 HIV-1 because patients with X4 HIV-1 progress to AIDS more rapidly. In contrast, all three nef genes studied here were derived from patient ACH142, who never developed X4 HIV-1 [60, 61]. More analyses of nef genes from R5 HIV-1 clones derived from progressors are needed to test the generality of our observations.
Previous studies have shown that X4 HIV-1 isolates are more sensitive to neutralization by soluble CD4 than R5 HIV-1 [62–64] and that X4 HIV-1 clones incorporate less Env into their virions when cellular CD4 is not downmodulated than R5 HIV-1 clones . It is therefore likely that downmodulation of CD4 has a greater impact on X4 HIV-1 replication than on R5 HIV-1 replication. R5 HIV-1 Nef may therefore have a greater positive effect on viral replication by down-modulating cell surface MHC-I molecules and thereby protecting infected cells from lysis by anti-HIV-1 cytotoxic T lymphocytes.
Moreover, because R5 HIV-1 clones preferentially infect effector memory T cells and macrophages while X4 HIV-1 clones preferentially infect naïve T cells, nef genes in each type of HIV-1 may evolve over the course of an infection to better enhance replication in each respective cell type. Determining whether such an evolution occurs may allow us to find specific Nef interactions that occur preferentially in macrophages or in memory or naïve T cells. In particular, Nef's ability to activate T cells may be more essential for X4 HIV-1, since naïve T cells are less easily activated than memory T cells.
Our data lend support to the notion that Nef cannot evolve over the course of disease to enhance all of its functions. This is true for our study in which an AIDS-associated Nef was reduced in its ability to downmodulate CD4 and enhanced in its ability to downmodulate MHC I and is also true for other studies where the reverse was found. This supports a paradigm whereby increased Nef mediated downmodulation of CD4 or MHC Class I molecules correlates with a loss in the other function. This paradigm may be created by structural constraints that limit the ability of Nef to perform both functions optimally or by competition for limiting cellular factors necessary for both processes. Determination of these constraints and/or factors will shed light on the role of Nef in HIV-1 replication and pathogenesis.
This work was supported by R01 AI47729, R01 AI34721 and R01 AI47008 awarded by the Division of AIDS, NIAID, NIH to DC, M-LH and DR respectively. KCO was partially supported by an Infectious Diseases Training Grant, T32 A107046, awarded to the University of Virginia
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