Summary Herpesviruses, including human cytomegalovirus (HCMV), encode multiple microRNAs (miRNA) whose targets are just being uncovered. Moreover, miRNA function during the virus life cycle is relatively unknown. We find that HCMV miRs UL112-1, US5-1, and US5-2 target multiple components of the host secretory pathway, including VAMP3, RAB5C, RAB11A, SNAP23, and CDC42. A HCMV miR UL112-1, US5-1, and US5-2 triple mutant displayed aberrant morphogenesis of the virion assembly compartment (VAC), increased secretion of non-infectious particles, and increased IL-6 release from infected cells. Ectopic expression of UL112-1, US5-1, and US5-2 or siRNAs directed against RAB5C, RAB11A, SNAP23, and CDC42 caused the loss of Golgi stacks with reorganization into structures that resemble the VAC and a decrease in cytokine release. These observations indicate that multiple HCMV miRNAs coordinately regulate reorganization of the secretory pathway to control cytokine secretion and facilitate formation of the VAC for efficient infectious virus production.

Highlights d Latent HCMV infection of stem cells induces the myelosuppressive cytokine TGF-b d HCMV miR-US5-2 targets the transcriptional repressor NAB1 to mediate TGF-b expression d HCMV miR-UL22A downregulates SMAD3 to block TGF-b signaling in the infected cell d Blocking TGF-b signaling is critical for HCMV latency and genome maintenance

Simian immunodeficiency virus (SIV) insert–expressing, 68-1 rhesus cytomegalovirus (RhCMV/SIV) vectors elicit major histocompatibility complex E (MHC-E)– and MHC-II–restricted, SIV-specific CD8+ T cell responses, but the basis of these unconventional responses and their contribution to demonstrated vaccine efficacy against SIV challenge in the rhesus monkeys (RMs) have not been characterized. We show that these unconventional responses resulted from a chance genetic rearrangement in 68-1 RhCMV that abrogated the function of eight distinct immunomodulatory gene products encoded in two RhCMV genomic regions (Rh157.5/Rh157.4 and Rh158-161), revealing three patterns of unconventional response inhibition. Differential repair of these genes with either RhCMV-derived or orthologous human CMV (HCMV)–derived sequences (UL128/UL130; UL146/UL147) leads to either of two distinct CD8+ T cell response types—MHC-Ia–restricted only or a mix of MHC-II– and MHC-Ia–restricted CD8+ T cells. Response magnitude and functional differentiation are similar to RhCMV 68-1, but neither alternative response type mediated protection against SIV challenge. These findings implicate MHC-E–restricted CD8+ T cell responses as mediators of anti-SIV efficacy and indicate that translation of RhCMV/SIV vector efficacy to humans will likely require deletion of all genes that inhibit these responses from the HCMV/HIV vector.