nef Gene Is Required for Robust Productive Infection by Simian Immunodeficiency Virus of T-Cell-Rich Paracortex in Lymph Nodes

The importance of the nef gene of simian immunodeficiency virus (SIV) for persistent active viral replication has been demonstrated in a macaque AIDS model (26). Defects in the nef gene not only lowered the magnitude of SIV infection but also allowed the host immune system to induce protective immunity against pathogenic SIVs (12, 24, 25). Findings of defective nef alleles in human immunodeficiency virus (HIV) isolates from infected individuals who have been categorized as long-term nonprogressors were the driving force behind studies on protective immunity against the AIDS virus (28). Based on those studies, the nef gene is generally accepted to play a key role in the pathogenesis of HIV/SIV (primate AIDS virus) infection (13). The functions of the nef gene in primate AIDS virus replication in vitro or ex vivo have been reported; they include enhancement of viral infectivity (20, 33), mediation of T-cell activation (4, 15, 32, 48), and down-regulation of cell surface molecules such as CD4 (1, 18), major histocompatibility complex (MHC) class I (21, 30), and CD28 in CD4+ T cells (52). However, the functions of the nef gene in the primate AIDS virus in vivo still remain unclear (for reviews, see references 17, 25, and 41). The importance of early events in AIDS virus infection in terms of viral replication, host immune response, and disease progression has been reported from HIV type 1 (HIV-1) clinical studies (45) and studies of animal AIDS models (34). In particular, due to considerations of feasibility in study design, early events of SIV infection in macaques were extensively investigated by examination of various tissues, viral strains, and infection routes (9, 10, 23, 29, 39, 44, 50, 51, 54-57). Reimann et al., using SIVmac251, reported that SIV-infected cells localized predominantly in T-cell-rich extrafollicular regions in lymph nodes (LNs) at primary infection (44). Lackner et al. performed extensive analyses of the spleen and thymus and reported similar results for SIVmac239 infection. They also found that cells infected with an attenuated strain, SIVmac1A11, were localized in follicles (29). The results of Chakrabarti et al. with SIVmac251 were relatively consistent with those of Reimann et al. and Lackner et al., but they also noted SIV+ cells scattered in the cortex (corresponding to follicles) at day 4 postinfection (p.i.) (9). Chakrabarti et al. found productive infection by a SIVmac251 nef mutant in germinal centers (GC) at day 7 p.i. and subsequent trapping of SIV virions in GC at day 15 p.i. (10). Stahl-Henning et al. examined localization of SIV-infected cells in tonsils at day 4 p.i. for macaques infected with SIVmac251 by an oral mucosal route (50); they found that whereas SIV-infected cells were observed in all compartments of the tonsils, the majority were CD4+ T cells in extrafollicular lymphoid tissue equivalent to the paracortex in LNs. Taken together, the results from these five studies show that pathogenic SIVmac251 and SIVmac239 infection occurred in both the T-cell-rich paracortex and follicles and that Δnef mutants and an attenuated strain, SIVmac1A11, replicated in follicles in LNs. Thus, early events after SIV infection are assumed to determine the outcome of AIDS virus infection, that is, whether the infected host controls the infection or not. Furthermore, we assumed that the nef gene plays an important role in determining these early events after primate AIDS virus infection. In this study, to clarify the role of the nef gene in SIV replication in vivo, we infected rhesus macaques with a molecularly cloned SIVmac239 with a functional nef gene as a wild-type virus or with its nef deletion mutant (Δnef). SIVmac239 infection induced AIDS in two of the three animals by 3 years p.i., and Δnef mutant infection was suppressed to low viral loads without any manifestation associated with AIDS except a gradual decline in naive CD4 T cells. We examined SIV infection in the LNs at weeks 2, 8, 72, and 82 p.i. At the peak of the primary infection, SIV-infected cells were observed in different locations in the LNs: for the SIVmac239-infected animals, most of the infected cells were localized in the T-cell-rich paracortex, whereas for the Δnef-infected animals, most were localized in B-cell-rich follicles. Our in vivo study indicated that the nef gene enhances SIV replication by robust productive infection in memory CD4+ T cells in the T-cell-rich paracortex in lymphoid tissues.