GENETIC VARIATION IN THE NEF GENES OF ISOLATES FROM MACAQUES INFECTED WITH MOLECULAR CLONED SIV/MNE

Abstract

Human immunodeficiency virus type one and type two (HIV-1 and HIV-2), and simian immunodeficiency virus are members of the lentivirus subfamily of retroviruses. All retroviruses contain gag, poi, and env genes. HIV and SIV have in addition to the genes common to all retroviruses, a number of accessory genes; vif, vpr, vpu (HIV-1), vpx (HIV-2 and SIV), tat, rev and nef Nef is a 27 kDa (HIV-1) or 32 kDa (HIV-2 and SIV) myristylated protein localized in the cytoplasmic face of the plasma membrane, and is encoded by an open reading frame located at the 3' end of the viral genome overlapping the U3 region of the 3' LTR. Conservation of this coding region among all HIV-1 and HIV-2 strains as well as among all the SIVs suggests that nef plays a role in natural infection. The role of nef in the viral life cycle is not well defined. Contradictions regarding nef function may be due to HIV-I and SIV isolates propagated in vitro which encode prematurely terminated and most likely nonfunctional Nef proteins as well as a significant degree of amino acid sequence variation between nef isolates both in vitro and in vivo. Part of the difficulty in studying the effect of HIV variation on the progression of AIDS is that the exact sequence of the infecting virus is not known, and sequences analyzed after the propagation of virus in cell culture do not represent the viruses present in the host. Therefore, an SIV model system in which variation can be measured in vivo using a defined molecular clone of known sequence was used in this study to follow genetic variation of the original clone over time in infected macaques. Genomic DNA was extracted from peripheral blood lymphocytes (PBLs) obtained at two different time points, early and late, postinfection from macaques inoculated with the biological clone SIV/Mne EllS. SIV nef sequences were amplified by nested polymerase chain reaction (PCR) to look directly for SIV variants without any of the selection imposed by cell culture. The PCR product was cloned into the pCR II vector, and several clones were sequenced. To analyze variation in the nef sequence, the sequences were aligned and compared to the SIV/Mne cl 8 sequence, a molecular clone from a library of genomic DNA from the EllS cell line. In general, there were many random and sporadic mutations seen throughout the sequences analyzed, but there seemed to be a strong selection for certain amino acid changes. In particular, a set of striking mutations reestablishing the consensus sequence derived from the sooty mangabey/HIV-2 group of viruses were seen in any of the sequences examined, especially at the later time points postinfection. In addition, regions of nef that might be important for its function were mostly conserved. Most of the sequences analyzed had a conserved myristylation signal, our stretches of residues that have been shown to be highly conserved among the nef sequences of the SIVs as well as the HIVs, a region with an acidic charge, a proline-rich repeat, and a potential protein kinase C phosphorylation site.