Small antisense RNAs targeted to the HIV-1 promoter have been shown to remodel the surrounding chromatin to a state unfavorable for transcriptional activation, yet transcriptional gene silencing (TGS) of HIV-1 has, to date, not been shown in primary human cells. We demonstrate here that TGS can reduce viral transcription in primary human CD4⁺ T cells; however, increasing viral burden results in the loss of this antiviral effect. This observation suggests a critical level at which viral RNA can dilute out effective targeting by TGS-based RNAs. Furthermore, studies into off-target effects have identified a potential interaction between the small nucleolar RNA pathway and the TGS-based antisense RNA, resulting in activation of p53. Although not overtly toxic to primary cells, this represents a novel interaction between antisense RNAs and a cellular pathway that should be considered when pursuing small antisense RNA-based therapeutics.