Programmed -1 ribosomal frameshifting is widely used in the expression of RNA virus replicases and represents a potential target for antiviral intervention. There is interest in determining the extent to which frameshifting efficiency can be modulated before virus replication is compromised, and we have addressed this question using the alpharetrovirus Rous sarcoma virus (RSV) as a model system. In RSV, frameshifting is essential in the production of the Gag-Pol polyprotein from the overlapping gag and pol coding sequences. The frameshift signal is composed of two elements, a heptanucleotide slippery sequence and, just downstream, a stimulatory RNA structure that has been proposed to be an RNA pseudoknot. Point mutations were introduced into the frameshift signal of an infectious RSV clone, and virus replication was monitored following transfection and subsequent infection of susceptible cells. The introduced mutations were designed to generate a range of frameshifting efficiencies, yet with minimal impact on encoded amino acids. Our results reveal that point mutations leading to a 3-fold decrease in frameshifting efficiency noticeably reduce virus replication and that further reduction is severely inhibitory. In contrast, a 3-fold stimulation of frameshifting is well tolerated. These observations suggest that small-molecule inhibitors of frameshifting are likely to have potential as agents for antiviral intervention. During the course of this work, we were able to confirm, for the first time in vivo, that the RSV stimulatory RNA is indeed an RNA pseudoknot but that the pseudoknot per se is not absolutely required for virus viability.