Although most of the attention was focused on the characterization of changes in the Spike protein among variants of SARS-CoV-2 virus, mutations outside the Spike region are likely to contribute to virus pathogenesis, virus adaptation and escape to the immune system. Phylogenetic analysis of SARS-CoV-2 Omicron strains reveals that several virus sub-lineages could be distinguished, from BA.1 up to BA.5. Regarding BA.1, BA.2 and BA.5, several mutations concern viral proteins with antagonistic activity to the innate immune system, such as NSP1 (S135R), which is involved in mRNAs translation, exhibiting a general shutdown in cellular protein synthesis. Additionally, mutations and/or deletions in the ORF6 protein (D61L) and in the nucleoprotein N (P13L, D31-33ERS, P151S, R203K, G204R and S413R) have been reported, although the impact of such mutations on protein function has not been further studied. The aim of this study was to better investigate the innate immunity modulation by different Omicron sub-lineages, in the attempt to identify viral proteins that may affect virus fitness and pathogenicity. Our data demonstrated that, in agreement with a reduced Omicron replication in Calu-3 human lung epithelial cells compared to the Wuhan-1 strain, a lower secretion of interferon beta (IFN-β) from cells was observed in all sub-lineages, except for BA.2. This evidence might be correlated with the presence of a mutation within the ORF6 protein (D61L), which is strikingly associated to the antagonistic function of the viral protein, since additional mutations in viral proteins acting as interferon antagonist were not detected or did not show significant influence. Indeed, the recombinant mutated ORF6 protein failed to inhibit IFN-β production in vitro. Furthermore, we found an induction of IFN-β transcription in BA.1 infected cells, that was not correlated with the cytokine release at 72 h post-infection, suggesting that post-transcriptional events can be involved in controlling the innate immunity.