Functional domains of the influenza A virus PB2 protein:identification of NP- and PB1-binding sites

AbstractInfluenza virus genomic RNA segments are packaged into ribonucleoprotein (RNP) structures by the PB1, PB2, and PA subunits of an RNA polymerase and a single-strand RNA-binding nucleoprotein (NP). Assembly and function of these ribonucleoproteins depend on a complex set of protein–protein and protein–RNA interactions. Here, we identify new functional domains of PB2. We show that PB2 contains two regions that bind NP and also identify a novel PB1 binding site. The regions of PB2 responsible for binding NP and PB1 show considerable overlap, and binding of NP to the PB2 fragments could be outcompeted by PB1. The binding domains of PB2 acted as trans-dominant inhibitors of viral gene expression, and consistent with the in vitro binding data, their inhibitory activity depended on the concentration of wild-type PB2, NP, and PB1. This provides evidence for functionally significant and potentially regulatory interactions between PB2 and NP

Sleepless latency of human cytomegalovirus.

As with all human herpesviruses, human cytomegalovirus (HCMV) persists for the lifetime of the host by establishing a latent infection, which is broken by periodic reactivation events. One site of HCMV latency is in the progenitor cells of the myeloid lineage such as CD34+ cells and their CD14+ derivatives. The development of experimental techniques to isolate and culture these primary cells in vitro is enabling detailed analysis of the events that occur during virus latency and reactivation. Ex vivo differentiation of latently infected primary myeloid cells to dendritic cells and macrophages results in the reactivation of latent virus and provides model systems in which to analyse the viral and cellular functions involved in latent carriage and reactivation. Such analyses have shown that, in contrast to primary lytic infection or reactivation which is characterised by a regulated cascade of expression of all viral genes, latent infection is associated with a much more restricted viral transcription programme with expression of only a small number of viral genes. Additionally, concomitant changes in the expression of cellular miRNAs and cellular proteins occur, and this includes changes in the expression of a number of secreted cellular proteins and intracellular anti-apoptotic proteins, which all have profound effects on the latently infected cells. In this review, we concentrate on the effects of one of the latency-associated viral proteins, LAcmvIL-10, and describe how it causes a decrease in the cellular miRNA, hsa-miR-92a, and a concomitant upregulation of the GATA2 myeloid transcription factor, which, in turn, drives the expression of cellular IL-10. Taken together, we argue that HCMV latency, rather than a period of viral quiescence, is associated with the virally driven manipulation of host cell functions, perhaps every bit as complex as lytic infection. A full understanding of these changes in cellular and viral gene expression during latent infection could have far-reaching implications for therapeutic intervention.We thank the MRC for funding, Grant Number G0701279. This research was supported by the Cambridge NIHR BRC cell phenotyping hub.This is the final published version. It first appeared at http://link.springer.com/article/10.1007%2Fs00430-015-0401-6

Understanding HCMV Latency Using Unbiased Proteomic Analyses

Human cytomegalovirus (HCMV) establishes either a latent (non-productive) or lytic (productive) infection depending upon cell type, cytokine milieu and the differentiation status of the infected cell. Undifferentiated cells, such as precursor cells of the myeloid lineage, support a latent infection whereas terminally differentiated cells, such as monocytes or dendritic cells are an environment conducive to reactivation and support a lytic infection. The mechanisms which regulate HCMV in either a latent or lytic infection have been the focus of intense investigation with a view to developing novel treatments for HCMV-associated disease which can have a heavy clinical burden after reactivation or primary infection in, especially, the immune compromised. To this end, a number of studies have been carried out in an unbiased manner to address global changes occurring within the latently infected cell to address the molecular changes associated with HCMV latency. In this review, we will concentrate on the proteomic analyses which have been carried out in undifferentiated myeloid cells which either stably express specific viral latency associated genes in isolation or on cells which have been latently infected with virus

Editorial: Cytomegalovirus Pathogenesis and Host Interactions.

Funding Information: MN and EP are funded by the Medical Research Council (MR/ P022146/1 and MR/S00081X/1, respectively).Publisher PDFPeer reviewe

GeoSocial: exploring the usefulness of social media mining in the applied natural geohazard sciences

Obtaining real-time information about a geohazard event as it unfolds, such as a flood or earthquake, used to be largely limited to the professional media. Nowadays, obtaining news stories from social media (e.g. Facebook, Twitter, YouTube, Flickr etc.), directly as they unfold, is becoming the ‘norm’ for many in society. The Haitian Earthquake in January 2010 and the Great East Japan Earthquake in March 2011, provided some of the first natural hazard examples, to really demonstrate the power of social media over traditional news sources for obtaining, live information from which people and authorities could gain situational awareness

Latent infection of myeloid progenitors by human cytomegalovirus protects cells from FAS-mediated apoptosis through the cellular IL-10/PEA-15 pathway.

Latent infection of primary CD34(+) progenitor cells by human cytomegalovirus (HCMV) results in their increased survival in the face of pro-apoptotic signals. For instance, we have shown previously that primary myeloid cells are refractory to FAS-mediated killing and that cellular IL-10 (cIL-10) is an important survival factor for this effect. However, how cIL-10 mediates this protection is unclear. Here, we have shown that cIL-10 signalling leading to upregulation of the cellular factor PEA-15 mediates latency-associated protection of CD34(+) progenitor cells from the extrinsic death pathway.We gratefully acknowledge funding from the UK Medical Research Council (J.H.S. G:0701279) which supports the current research in our laboratory and also the support of NIHR UK Biomedical Research Centre (J.H.S.). We thank Linda Teague, Roy Whiston and Stuart McGregor Dallas for technical support and Stuart McGregor Dallas for providing validation data for figure 1.This is the final version. It first appeared at http://jgv.sgmjournals.org/content/journal/jgv/10.1099/vir.0.000180

Depletion of cellular pre-replication complex factors results in increased human cytomegalovirus DNA replication.

Although HCMV encodes many genes required for the replication of its DNA genome, no HCMV-encoded orthologue of the origin binding protein, which has been identified in other herpesviruses, has been identified. This has led to speculation that HCMV may use other viral proteins or possibly cellular factors for the initiation of DNA synthesis. It is also unclear whether cellular replication factors are required for efficient replication of viral DNA during or after viral replication origin recognition. Consequently, we have asked whether cellular pre-replication (pre-RC) factors that are either initially associated with cellular origin of replication (e.g. ORC2), those which recruit other replication factors (e.g. Cdt1 or Cdc6) or those which are subsequently recruited (e.g. MCMs) play any role in the HCMV DNA replication. We show that whilst RNAi-mediated knock-down of these factors in the cell affects cellular DNA replication, as predicted, it results in concomitant increases in viral DNA replication. These data show that cellular factors which initiate cellular DNA synthesis are not required for the initiation of replication of viral DNA and suggest that inhibition of cellular DNA synthesis, in itself, fosters conditions which are conducive to viral DNA replication

Brief Report: Which Came First? Exploring Crossmodal Temporal Order Judgements and Their Relationship with Sensory Reactivity in Autism and Neurotypicals

Previous studies have indicated that visual-auditory temporal acuity is reduced in children with autism spectrum conditions (ASC) in comparison to neurotypicals. In the present study we investigated temporal acuity for all possible bimodal pairings of visual, tactile and auditory information in adults with ASC (n = 18) and a matched control group (n = 18). No group differences in temporal acuity for crossmodal stimuli were observed, suggesting that this may be typical in adults with ASC. However, visual-tactile temporal acuity and bias towards vision when presented with visual-auditory information were both predictors of self-reported sensory reactivity. This suggests that reduced multisensory temporal acuity and/or attention towards vision may contribute to atypical sensory reactivity

The immunology of human cytomegalovirus latency: could latent infection be cleared by novel immunotherapeutic strategies?

While the host immune response following primary human cytomegalovirus (HCMV) infection is generally effective at stopping virus replication and dissemination, virus is never cleared by the host and like all herpesviruses, persists for life. At least in part, this persistence is known to be facilitated by the ability of HCMV to establish latency in myeloid cells in which infection is essentially silent with, importantly, a total lack of new virus production. However, although the viral transcription programme during latency is much suppressed, a number of viral genes are expressed during latent infection at the protein level and many of these have been shown to have profound effects on the latent cell and its environment. Intriguingly, many of these latency-associated genes are also expressed during lytic infection. Therefore, why the same potent host immune responses generated during lytic infection to these viral gene products are not recognized during latency, thereby allowing clearance of latently infected cells, is far from clear. Reactivation from latency is also a major cause of HCMV-mediated disease, particularly in the immune compromised and immune naive, and is also likely to be a major source of virus in chronic subclinical HCMV infection which has been suggested to be associated with long-term diseases such as atherosclerosis and some neoplasias. Consequently, understanding latency and why latently infected cells appear to be immunoprivileged is crucial for an understanding of the pathogenesis of HCMV and may help to design strategies to eliminate latent virus reservoirs, at least in certain clinical settings.This work was funded by British Medical Research Council Grant JS and MW - G0701279 and MR/K021087/1 and supported by the NIHR Cambridge BRC Cell Phenotyping hub.This is the accepted manuscript. The final version is available from the publisher at http://www.nature.com/cmi/journal/vaop/ncurrent/full/cmi201475a.html