The simian-human immunodeficiency virus (SHIV)/ macaque model for human immunodeficiency virus type 1 has become a useful tool to assess the role of Vpu in lentivirus pathogenesis. In this report, we have mutated the two phosphorylated serine residues of the HIV-1 Vpu to glycine residues and have reconstructed a SHIV expressing this nonphosphorylated Vpu (SHIV(S52,56G)). Expression studies revealed that this protein was localized to the same intracellular compartment as wild-type Vpu. To determine if this virus was pathogenic, four pig-tailed macaques were inoculated with SHIV(S52,56G) and virus burdens and circulating CD4(+) T cells monitored up to 1 year. Our results indicate that SHIV(S52,56G) caused rapid loss in the circulating CD4(+) T cells within 3 weeks of inoculation in one macaque (CC8X), while the other three macaques developed no or gradual numbers of CD4(+) T cells and a wasting syndrome. Histological examination of tissues revealed that macaque CC8X had lesions in lymphoid tissues (spleen, lymph nodes, and thymus) that were typical for macaques inoculated with pathogenic parental SHIV(KU-1bMC33) and had no lesions within the CNS. To rule out that macaque CC8X had selected for a virus in which there was reversion of the glycine residues at positions 52 and 56 to serine residues and/or compensating mutations occurred in other genes associated with CD4 down-regulation, sequence analysis was performed on amplified vpu sequences isolated from PBMC and from several lymphoid tissues at necropsy. Sequence analysis revealed a reversion of the glycine residues back to serine residues in this macaque. The other macaques maintained low virus burdens, with one macaque (P003) developing a wasting syndrome between months 9 and 11. Histological examination of tissues from this macaque revealed a thymus with severe atrophy that was similar to that of a previously reported macaque inoculated with a SHIV lacking vpu (Virology 293, 2002, 252). Sequence analysis revealed no reversion of the glycine residues in the vpu sequences isolated from this macaque. These results contrast with those from four macaques inoculated with the parental pathogenic SHIV(KU-1bMC33), all of which developed severe CD4(+) T cell loss within 1 month after inoculation. Taken together, these results indicate that casein kinase II phosphorylation sites of Vpu contributes to the pathogenicity of the SHIV(KU-1bMC33) and suggest that the SHIV(KU-1bMC33)/pig-tailed macaque model will be useful in analyzing amino acids/domains of Vpu that contribute to the pathogenesis of HIV-1.