Cytomegalovirus (CMV) can super-infect persistently infected hosts despite CMV-specific humoral and cellular immunity; however, how it does so remains undefined. Here, we demonstrate that super-infection of rhesus CMV-infected rhesus macaques (RM) requires evasion of CD8+ T cell immunity by virally-encoded inhibitors of MHC-I antigen presentation, particularly the homologues of human CMV US2, 3, 6 and 11. In contrast, MHC-I interference was dispensable for primary infection of RM, or for the establishment of a persistent secondary infection in CMV-infected RM transiently depleted of CD8+ lymphocytes. These findings demonstrate that US2-11 glycoproteins promote evasion of CD8+ T cells in vivo thus supporting viral replication and dissemination during super-infection, a process that complicates the development of preventative CMV vaccines, but that can be exploited for CMV-based vector development.

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SUMMARY Microglia are the intrinsic immune sentinels of the central nervous system. Their activation restricts tissue injury and pathogen spread, but in some settings, including viral infection, this response can contribute to cell death and disease. Identifying mechanisms that control microglial responses is therefore an important objective. Using replication-incompetent adenovirus 5 (Ad5)-based vectors as a model, we investigated the mechanisms through which microglia recognize and respond to viral uptake. Transgenic, immunohistochemical, molecular-genetic, and fluorescence imaging approaches revealed that phosphatidylserine (PtdSer) exposure on the outer leaflet of transduced cells triggers their engulfment by microglia through TAM receptor-dependent mechanisms. We show that inhibition of phospholipid scramblase 1 (PLSCR1) activity reduces intracellular calcium dysregulation, prevents PtdSer externalization, and enables months-long protection of vector-transduced, transgene-expressing cells from microglial phagocytosis. Our study identifies PLSCR1 as a potent target through which the innate immune response to viral vectors, and potentially other stimuli, may be controlled.

Summary The evolution of minimal DNA tumor virus’ genomes has selected for small oncoproteins that hijack critical cellular protein interaction networks. The structural basis for the dominant interactions of adenovirus oncoproteins has remained elusive as no full-length structures have been solved. Adenovirus E4-ORF3 forms a nuclear scaffold and simultaneously inactivates p53, PML, TRIM24 and MRE11/RAD50/NBS1 (MRN) tumor suppressors. We identify oligomerization mutants and solve the crystal structure of an E4-ORF3 dimer. The structure of E4-ORF3 is unrelated to known polymers or oncogenes. E4-ORF3 dimers have a central β-core and co-assemble in multiple configurations by exchanging their C-terminal tails. The resulting polymers are disordered weaves of linear and branched oligomer threads that form a 3D network that partitions the nucleus. The assembly of E4-ORF3 creates avidity driven interactions with PML and an emergent MRN binding interface. This study unveils an elegant solution whereby a small domain forms a multivalent polymer to disrupt multiple protein complexes.

Human CMV is the predominant infectious cause of congenital birth defects and an opportunistic pathogen in immunosuppressed individuals, including AIDS patients. Most individuals are infected early during their life followed by life-long latent infection. During this latent phase, frequent reactivation and antigen production continue to stimulate the immune system. While the immune response is able to control the virus, it is unable to eradicate it. Moreover, super-infection by diVerent CMV strains has been observed despite a strong immune response. Longterm immune stimulation by CMV has also been implicated in immune senescence and chronic conditions such as atherosclerosis. CMVs are highly species-speciWc and the relatedness of CMV genomes exactly mirrors the relatedness of their hosts. Thus, each CMV species is highly adapted to its respective host species, but is unable to infect other, even closely related hosts. While fascinating from an evolutionary perspective, this host restriction prevents studying HCMV in experimental animals. Exceptions are severely immunocompromised mice, e.g. SCID mice, or SCID/NOD mice, which might allow partial reconstitution of CMV infection in rodents. More practical however, is to study CMVs in their natural host, e.g. murine, rat or guinea pig CMVs. However, while these small animal models have many advantages, such as the availability of inbred animals as well as lower cost, the limited homology of the viral genomes with HCMV limits the functional analysis of homologous gene products. The closest relative to HCMV is chimpanzee CMV (CCMV), but this is not a practical animal model since chimps are a protected species, extremely expensive and of very limited availability. In contrast, rhesus macaques are a more widely used experimental animal species and, while more distant than CCMV, rhesus CMV (RhCMV) contains most of the HCMV gene families thus allowing the study of their role in acute and latent CMV infection. In this review we will discuss the current state of developing RhCMV as a model for HCMV.