Grouppe Kurosawa: HIV - The Biology of NEF (print)

The Biology of NEF

95% of NEF is located in the cytoplasm and nucleus, while the final 5% is associated with the plasma membrane. Five days after an acute HIV infection, 90% of infected cells express NEF in the cytoplasm, while only 20% express GAG and ENV. NEF is myristylated which enables it to interact with lipid membranes and cytoskeletal proteins. However, in order for NEF to interact with the plasma membrane, the NEF protein must be expressed by a retroviral vector. The implication is that another retroviral protein, possibly a protease, is required to help NEF integrate into the plasma membrane. When NEF was associated with the plasma membrane, it activated early signaling events associated with the T cell antigen receptor, i.e. the expression of CD69 and the IL2R, and a 10-20 fold increase in the transcription of the HIV provirus. NEF also induced a state of apoptosis or programmed cell death in a high percentage of the activated cells. Cytoplasmic NEF inhibited early signaling events associated with the T cell receptor possibly by binding and sequestering activation factors away from the plasma membrane. An additional interpretation is that NEF interacts with a cytoplasmic protein that directly or indirectly inhibits the induction of activation factors such as NF-kB and AP-1. In summary, in vitro NEF activates or inhibits viral transcription depending on its cellular localization while in vivo NEF is absolutely necessary for the maintenance of a high viral titer and the progression to AIDS. Although paradoxical, the in vitro and in vivo effects of NEF are not necessarily contradictory.

Much of the in vivo work on NEF has been conducted with NEF-deleted mutants (NEF minus) of SIV, or simian immunodeficiency virus. This virus is very similar to HIV, and the biology of its gene products can be studied relatively easily in New World monkeys (marmosets, macaques). SIV strains with NEF deletions are not pathogenic when injected into monkeys. Although in vitro infectivity studies showed no differences in the replication of wild-type and mutant virus, 100 fold more lymphocytes were required from infected monkeys to rescue NEF deleted viruses than were necessary for wild-type virus. Clearly, in vivo, NEF positively influences the viral titer, but this does imply a direct effect on transcription. NEF minus viral strains may actually replicate faster in vivo, and generate a more vigorous immune response against virally infected cells. Wild-type virus, as in vitro studies have already shown, may actually repress viral transcription, thereby inducing a form of defacto viral latency in infected cells. Months after infection, the NEF minus strains may have already been cleared from the body. In contrast, the high viral titers associated with NEF positive strains may be the result of the virus's ability to impair the immune response to its very presence. Monkeys infected with high doses of NEF negative virus induce a vigorous immune response against the virus, which protected the monkeys against subsequent challenge with wild-type, pathogenic strains. Since these viruses differed only by the presence or absence of the NEF gene, NEF may induce a state of immunosuppression or dysfunction in the body.

The amino terminal domain of NEF is both myristylated and fusogenic for small phosphorylated lipid vesicles. The fusogenic peptide has a striking sequence homology to melittin, a bee venom toxin that is known to induce phase changes in phospholipid membranes. NEF is released from both infected and transfected cells, and this release is dependent upon the presence of the first 18 amino acids of the protein. Extracellular NEF may play a significant pathogenic role in the development of AIDS because it activates productive HIV-1 infections from a state of latency, and stimulates the maturation of B cells and release of non-specific antibody. Hypergammaglobulinemia is an early diagnostic sign in the development of AIDS, and may account for the production and release of inappropriate, low affinity antibody and the development of autoimmune phenomenon. NEF is expressed in the membrane as an integral membrane protein, which has a number of its carboxyl-terminal domain sites exposed to the extracellular environment. These NEF domains can interact with uninfected CD4 T cells, resulting in cellular fusion and the induction of apoptosis in both cells. Since NEF is expressed in infected cells before the transcription of envelope and structural proteins, the interaction of cell surface NEF with uninfected CD4 T cells may precede that of gp120 with CD4.

A neutralizing immune response to NEF can take many forms. ADCC antibodies directed against the extracellular NEF domains expressed on the membranes of infected cells could activate natural killer cells and cytotoxic monocytes to kill the cells harboring the virus. Neutralizing antibodies could additionally prevent NEF, usually secreted in the membranes of phospholipid vesicles, from infecting new CD4 T cells, while cytotoxic T cells could recognize NEF peptides associated with class I histocompatibility antigens. Since NEF increases the efficacy of reverse transcriptase in newly infected cells, NEF may be a cell membrane component of secreted virus, and play a role in the infectivity of new cells. This has not been conclusively established. However, this argument is strengthened by NEF's relative insolubility in aqueous solutions, and the observation that NEF was secreted associated with phospholipid vesicles. Non-infected heterosexuals who have had repeated contact with HIV infected individuals have strong NEF-specific cytotoxic T cells in their blood as do macaques that have survived infection with pathogenic strains of SIV. NEF is an extremely immunogenic protein which, at low multiplicity's of infection, may play a fundament role in activating various arms of the immune system against HIV. At high multiplicity's of infection, as is often found in individuals whose conditions rapidly deteriorate towards AIDS, NEF may induce a state of immunosuppression or dysfunction before virus and virally infected cells can be cleared from the body. NEF, devoid of part of its amino terminal domain, is an important component of an HIV vaccine.