Viruses must enter host cells to replicate, assemble and propagate. Because of the restricted size of their genomes, viruses have had to evolve efficient ways of exploiting host cell processes to promote their own life cycles and also to escape host immune defence mechanisms. Many viral open reading frames (viORFs) with immune-modulating functions essential for productive viral growth have been identified across a range of viral classes. However, there has been no comprehensive study to identify the host factors with which these viORFs interact for a global perspective of viral perturbation strategies. Here we show that different viral perturbation patterns of the host molecular defence network can be deduced from a mass-spectrometry-based host-factor survey in a defined human cellular system by using 70 innate immune-modulating viORFs from 30 viral species. The 579 host proteins targeted by the viORFs mapped to an unexpectedly large number of signalling pathways and cellular processes, suggesting yet unknown mechanisms of antiviral immunity. We further experimentally verified the targets heterogeneous nuclear ribonucleoprotein U, phosphatidylinositol-3-OH kinase, the WNK (with-no-lysine) kinase family and USP19 (ubiquitin-specific peptidase 19) as vulnerable nodes in the host cellular defence system. Evaluation of the impact of viral immune modulators on the host molecular network revealed perturbation strategies used by individual viruses and by viral classes. Our data are also valuable for the design of broad and specific antiviral therapies.

SUMMARY Virus infection-induced global protein synthesis suppression is linked to assembly of stress granules (SGs), cytosolic aggregates of stalled translation preinitiation complexes. To study long-term stress responses, we developed an imaging approach for extended observation and analysis of SG dynamics during persistent hepatitis C virus (HCV) infection. In combination with type 1 interferon, HCV infection induces highly dynamic assembly/disassembly of cytoplasmic SGs, concomitant with phases of active and stalled translation, delayed cell division, and prolonged cell survival. Double-stranded RNA (dsRNA), independent of viral replication, is sufficient to trigger these oscillations. Translation initiation factor eIF2α phosphorylation by protein kinase R mediates SG formation and translation arrest. This is antagonized by the upregulation of GADD34, the regulatory subunit of protein phosphatase 1 dephosphorylating eIF2α. Stress response oscillation is a general mechanism to prevent long-lasting translation repression and a conserved host cell reaction to multiple RNA viruses, which HCV may exploit to establish persistence.

The processivity factor of the human cytomegalovirus (HCMV) DNA polymerase phosphoprotein ppUL44 plays an essential role in viral replication, showing nuclear localization in infected cells. The present study examines ppUL44's nuclear import pathway for the first time, ectopic expression of ppUL44 revealing a strong nuclear localization in transfected COS-7 and other cell types, implying that no other HCMV proteins are required for nuclear transportation and retention. We show that of the two potential nuclear localization signals (NLSs) located at amino acids 162-168 (NLS1) and 425-431 (NLS2), NLS2 is necessary and sufficient to confer nuclear localization. Moreover, using enzyme-linked immunosorbent assays and gel mobility shift assays, we show that NLS2 is recognized with high affinity by the importin (IMP) a/b heterodimer. Using gel mobility shift and transient transfection assays, we find that flanking sequences containing a cluster of potential phosphorylation sites, including a consensus site for protein kinase CK2 (CK2) at Ser 413 upstream of the NLS, increase NLS2-dependent IMP binding and nuclear localization, suggesting a role for these sites in enhancing UL44 nuclear transport. Results from site-directed mutagenic analysis and live-cell imaging of green fluorescent protein (GFP)-UL44 fusion protein-expressing cells treated with the CK2-specific inhibitor 4,5,6,7-tetrabromobenzotriazole are consistent with phosphorylation of Ser 413 enhancing ppUL44 nuclear transport.

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