Evidence for a crucial role of a host non-coding RNA in influenza A virus replication.

A growing body of evidence suggests the non-protein coding human genome is of vital importance for human cell function. Besides small RNAs, the diverse class of long non-coding RNAs (lncRNAs) recently came into focus. However, their relevance for infection, a major evolutionary driving force, remains elusive. Using two commercially available microarray systems, namely NCode™ and Sureprint™ G3, we identified differential expression of 42 ncRNAs during influenza A virus (IAV) infection in human lung epithelial cells. This included several classes of lncRNAs, including large intergenic ncRNAs (lincRNAs). As analyzed by qRT-PCR, expression of one lincRNA, which we termed virus inducible lincRNA (VIN), is induced by several IAV strains (H1N1, H3N2, H7N7) as well as vesicular stomatitis virus. However, we did not observe an induction of VIN by influenza B virus, treatment with RNA mimics, or IFNß. Thus, VIN expression seems to be a specific response to certain viral infections. RNA fractionation and RNA-FISH experiments revealed that VIN is localized to the host cell nucleus. Most importantly, we show that abolition of VIN by RNA interference restricts IAV replication and viral protein synthesis, highlighting the relevance of this lincRNA for productive IAV infection. Our observations suggest that viral pathogens interfere with the non-coding portion of the human genome, thereby guaranteeing their successful propagation, and that the expression of VIN correlates with their virulence. Consequently, our study provides a novel approach for understanding virus pathogenesis in greater detail, which will enable future design of new antiviral strategies targeting the host's non-protein coding genome.

Nucleosome maps of the human cytomegalovirus genome reveal a temporal switch in chromatin organization linked to a major IE protein.

Human CMV (hCMV) establishes lifelong infections in most of us, causing developmental defects in human embryos and life-threatening disease in immunocompromised individuals. During productive infection, the viral >230,000-bp dsDNA genome is expressed widely and in a temporal cascade. The hCMV genome does not carry histones when encapsidated but has been proposed to form nucleosomes after release into the host cell nucleus. Here, we present hCMV genome-wide nucleosome occupancy and nascent transcript maps during infection of permissive human primary cells. We show that nucleosomes occupy nuclear viral DNA in a nonrandom and highly predictable fashion. At early times of infection, nucleosomes associate with the hCMV genome largely according to their intrinsic DNA sequence preferences, indicating that initial nucleosome formation is genetically encoded in the virus. However, as infection proceeds to the late phase, nucleosomes redistribute extensively to establish patterns mostly determined by nongenetic factors. We propose that these factors include key regulators of viral gene expression encoded at the hCMV major immediate-early (IE) locus. Indeed, mutant virus genomes deficient for IE1 expression exhibit globally increased nucleosome loads and reduced nucleosome dynamics compared with WT genomes. The temporal nucleosome occupancy differences between IE1-deficient and WT viruses correlate inversely with changes in the pattern of viral nascent and total transcript accumulation. These results provide a framework of spatial and temporal nucleosome organization across the genome of a major human pathogen and suggest that an hCMV major IE protein governs overall viral chromatin structure and function.