Viral factors as well as host ones play major roles in the disease progression of human immunodeficiency virus type 1 (HIV-1) infection. We have examined cytotoxic T-lymphocyte activity and HIV-1 DNA PCR results of 312 high-risk seronegative drug users in northern Thailand and identified four seronegative cases positive for both assays. Furthermore, we have identified a synonymous mutation in nucleotide position 75 of the gag p17 gene (A426G) of HIV-1 that belongs to the CRF01_AE virus circulating in Thailand. The replication-competent HIV-1 clone containing the A426G mutation demonstrated a dramatic reduction of virion production and perturbation of viral morphogenesis without affecting viral protein synthesis in cells.