Antiviral Activity Does Not Establish Packaging Inhibition: An Evidence Audit of HIV-1 Packaging-Signal RNA Decoys

Background. Selective encapsidation of HIV-1 genomic RNA (gRNA) is essential for infectivity, and RNA constructs derived from the packaging signal (Ψ) have been explored as antivirals for over three decades. Antiviral activity and packaging-specific inhibition are, however, distinct claims, and the literature has not consistently separated them. Objective. To audit the published evidence for RNA-mediated interference with HIV-1 gRNA packaging, grading each study by whether packaging itself was measured and whether the observed phenotype could be assigned to packaging, and to reconcile the historical record with mechanistic work published through 2026. Methods. Targeted evidence audit using PubMed, PubMed Central and publisher full text, with backward citation chaining from primary intervention studies. Each study was classified by intervention architecture, presence of a direct packaging readout, and strength of mechanistic attribution. Not a systematic review or meta-analysis. Results. Genuine packaging-related positives exist: interfering RNAs containing an extended Ψ, the dimer linkage structure and RRE are co-packaged into progeny and reduce infectivity, and a SELEX-derived high-affinity Gag/NC ligand can substitute for SL3 in cis. But where packaging was measured directly, antiviral activity often failed to map cleanly onto packaging-specific inhibition. Ψ-transcript-expressing Jurkat lines showed prolonged replication resistance with unchanged gRNA packaging efficiency, an effect that extended to HIV-2 and did not correlate with decoy expression. A 5′-LTR decoy reduced p24 with no detectable packaging inhibition. Antisense constructs directed at the splice-donor/Ψ region inhibited replication, and a longer construct also reduced virion RNA packaging, but the authors attributed the predominant effect to earlier post-transcriptional steps. A 16-mer SL3 decoy delivered efficiently into lymphocytes produced only modest cytoprotection and no significant viral-load suppression. Three recent findings constrain interpretation further. Kinetic discrimination by Gag is an emergent property of multiple stem-loops and is undetectable for isolated stem-loops. Gag chimeras bearing heterologous RNA-binding domains of widely differing specificity all recruited gRNA to the plasma membrane, indicating that initial cytosolic recognition may not be the rate-limiting step. And Ψ binding shifts Gag toward the extended, assembly-competent conformation more than non-Ψ RNA does, raising the untested possibility that a Ψ-mimetic decoy is conformationally activating rather than purely sequestering. Conclusions. The record does not support a model in which a short Ψ-mimetic RNA reliably inhibits HIV-1 by competitively excluding gRNA from virions. A four-level interpretive ladder and a minimum reporting standard for future packaging-inhibition claims are proposed. Evidence cutoff: 15 September 2026.