SARS-CoV-2 has undergone rapid genetic diversification since its emergence in late 2019, giving rise to multiple lineages and variants of concern (VOCs). While many mutations in the viral Spike (S) protein affect antibody recognition and receptor binding, the contribution of non-S mutations to viral replication is less well understood. To address this, we generated a panel of recombinant variants of concern (rVOCs) expressing the same S:D614G gene. rAlpha, rBeta, rDelta, rGamma, rOmicron(BA.1) and rOmicron(BA.5) S:D614G replicated comparably to parental rSARS-CoV-2 S:D614G in VeroE6-TMPRSS2 (VTN) cells. In contrast, rBeta and rOmicron BA.5 S:D614G showed significantly attenuated replication in Calu-3 cells. Alpha, Beta and BA.5 S:D614G viruses formed significantly smaller plaques in VTN cells, suggesting that non-S lineage defining mutations alter cell-to-cell spread. Competition assays in Calu-3 cells demonstrated reduced fitness of rBeta and rOmicron BA.5 S:D614G, both of which were outcompeted by rSARS-CoV-2 S:D614G after a single passage. Attenuated replication of rBeta S:D614G was rescued by blocking JAK-STAT signalling with ruxolitinib. Transcriptomic analysis of Calu-3 cells infected with rBeta S:D614G +/- ruxolitinib revealed comparable induction of host innate antiviral response genes to rSARS-CoV-2 S:D614G at significantly lower RNA levels. Therefore, the interferon response is a major, but not the sole, barrier for replication of rBeta S:D614G in Calu-3 cells. The Beta, BA.1 and BA.5 S:D614G viruses replicated to significantly lower titres in the lung, nasal tissue and brain of K18-hACE2 mice than rSARS-CoV-2 S:D614G and did not cause significant weight loss. Transcriptomic analysis of murine lung tissue is currently in progress to establish if comparable transcription patterns are observed in vitro and in vivo. Together, these data highlight the importance of monitoring non-S lineage-defining mutations as viral variants emerge, as they can significantly alter viral replication phenotypes.