Alfalfa mosaic virus (AlMV) RNAs 1 and 2 with deletions in their 3' non‑translated regions (NTRs) have been previously shown to be encapsidated into virions by coat protein (CP) expressed from RNA3, indicating that the 3' NTRs of RNAs 1 and 2 are not required for virion assembly. Here, we constructed various mutants by deleting sequences within the 3' NTR of AlMV subgenomic (sg) RNA4 (same as of RNA3) and examined the effect of these deletions on replication and translation of chimeric Tobacco mosaic virus (TMV) expressing AlMV sgRNA4 from the TMV CP sg promoter (Av/A4) in tobacco protoplasts and Nicotiana benthamiana plants. While the Av/A4 mutants were as competent as the wild-type Av/A4 in RNA replication in protoplasts, their encapsidation, long-distance movement and virus accumulation varied significantly in N. benthamiana. These data suggest that the 3' NTR of AlMV sgRNA4 contains potential elements necessary for virus encapsidation.