Interferon beta (IFN-␤

During viral infections cellular gene expression is subject to rapid alterations induced by both viral and antiviral mechanisms. In this study, we applied metabolic labeling of newly transcribed RNA with 4-thiouridine (4sU-tagging) to dissect the real-time kinetics of cellular and viral transcriptional activity during lytic murine cytomegalovirus (MCMV) infection. Microarray profiling on newly transcribed RNA obtained at different times during the first six hours of MCMV infection revealed discrete functional clusters of cellular genes regulated with distinct kinetics at surprising temporal resolution. Immediately upon virus entry, a cluster of NF-κB- and interferon-regulated genes was induced. Rapid viral counter-regulation of this coincided with a very transient DNA-damage response, followed by a delayed ER-stress response. Rapid counter-regulation of all three clusters indicated the involvement of novel viral regulators targeting these pathways. In addition, down-regulation of two clusters involved in cell-differentiation (rapid repression) and cell-cycle (delayed repression) was observed. Promoter analysis revealed all five clusters to be associated with distinct transcription factors, of which NF-κB and c-Myc were validated to precisely match the respective transcriptional changes observed in newly transcribed RNA. 4sU-tagging also allowed us to study the real-time kinetics of viral gene expression in the absence of any interfering virion-associated-RNA. Both qRT-PCR and next-generation sequencing demonstrated a sharp peak of viral gene expression during the first two hours of infection including transcription of immediate-early, early and even well characterized late genes. Interestingly, this was subject to rapid gene silencing by 5–6 hours post infection. Despite the rapid increase in viral DNA load during viral DNA replication, transcriptional activity of some viral genes remained remarkably constant until late-stage infection, or was subject to further continuous decline. In summary, this study pioneers real-time transcriptional analysis during a lytic herpesvirus infection and highlights numerous novel regulatory aspects of virus-host-cell interaction.

The rabies virus (RV) phosphoprotein (P) is a type I interferon (IFN) antagonist preventing both transcriptional induction of IFN and IFN-mediated JAK/STAT signaling. In addition, P is an essential cofactor of the viral polymerase and is required for encapsidation of viral RNA into nucleoprotein during replication. By site-directed mutagenesis, we have identified a domain of P required for efficient inhibition of IFN induction. Phosphoproteins lacking amino acids (aa) 176 to 181, 182 to 186, or 176 to 186 were severely compromised in counteracting phosphorylation of IRF3 and IRF7 by TBK1 or IKKi while retaining the full capacity of preventing nuclear import of activated STATs and of supporting virus transcription and replication. Recombinant RV carrying the mutated phosphoproteins (the SAD ⌬Ind1, SAD ⌬Ind2, and SAD ⌬Ind1/2 viruses) activated IRF3 and beta IFN (IFN-␤) transcription in infected cells but still blocked STAT-mediated expression of IFN-stimulated genes. Due to a somewhat higher transcription rate, the SAD ⌬Ind1 virus activated IRF3 more efficiently than the SAD ⌬Ind2 virus. After intracerebral injection into mouse brains at high doses, the SAD ⌬Ind1 virus was completely apathogenic for wild-type (wt) mice, while the SAD ⌬Ind2 virus was partially attenuated and caused a slower progression of lethal rabies than wt RV. Neurovirulence of IFNresistant RV thus correlates with the capacity of the virus to prevent activation of IRF3 and IRF7.

The family Rhabdoviridae contains important pathogens of humans, livestock, and crops, including the insect-transmitted vesicular stomatitis virus (VSV) and the neurotropic rabies virus (RV), which is directly transmitted between mammals. In spite of a highly similar organization of RNA genomes, proteins, and virus particles, cell biology of VSV and RV is divergent in several aspects, particularly with respect to their interplay with the cellular host defense. While infection with both rhabdoviruses is recognized via viral triphosphate RNAs by the cytoplasmic RNA helicase/translocase RIG-I, the viral counteractions to limit the response are contrasting. VSV infection is characterized by a rapid general shutdown of host gene expression and severe cytopathic effects, due to multiple activities of the matrix (M) protein affecting host polymerase functions and mRNA nuclear export, and by rapid and high-level virus replication. In contrast, RV spread and transmission relies on preserving the integrity of host cells, particularly of neurons. While a general cell shutdown by RV M is not observed, RV phosphoprotein (P) has developed independent functions to interfere with activation of IRFs and with STAT signaling. The molecular mechanisms employed are different from those of the paramyxovirus P gene products serving similar functions, and illustrate evolution of IFN antagonists to specifically support virus survival in the natural niches.

No abstract