The human respiratory syncytial virus (HRSV) core viral RNA polymerase comprises the large polymerase protein (L) and its cofactor, the phosphoprotein (P), which associate with the viral ribonucleoprotein complex to replicate the genome and, together with the M2-1 protein, transcribe viral mRNAs. While cellular proteins have long been proposed to be involved in the synthesis of HRSV RNA by associating with the polymerase complex, their characterization has been hindered by the difficulty of purifying the viral polymerase from mammalian cell culture. In this study, enhanced green fluorescent protein (EGFP)-tagged L-and P-protein expression was coupled with high-affinity anti-GFP antibody-based immunoprecipitation and quantitative proteomics to identify cellular proteins that interacted with either the L-or the P-proteins when expressed as part of a biologically active viral RNP. Several core groups of cellular proteins were identified that interacted with each viral protein including, in both cases, protein chaperones. Ablation of chaperone activity by using small-molecule inhibitors confirmed previously reported studies which suggested that this class of proteins acted as positive viral factors. Inhibition of HSP90 chaperone function in the current study showed that HSP90 is critical for L-protein function and stability, whether in the presence or absence of the P-protein. Inhibition studies suggested that HSP70 also disrupts virus biology and might help the polymerase remodel the nucleocapsid to allow RNA synthesis to occur efficiently. This indicated a proviral role for protein chaperones in HRSV replication and demonstrates that the function of cellular proteins can be targeted as potential therapeutics to disrupt virus replication. IMPORTANCEHuman respiratory syncytial virus (HRSV) represents a major health care and economic burden, being the main cause of severe respiratory infections in infants worldwide. No vaccine or effective therapy is available. This study focused on identifying those cellular proteins that potentially interact specifically with the viral proteins that are central to virus replication and transcription, with a view to providing potential targets for the development of a specific, transient therapeutic which disrupts virus biology but prevents the emergence of resistance, while maintaining cell viability. In particular, protein chaperones (heat shock proteins 70 and 90), which aid protein folding and function, were identified. The mechanism by which these chaperones contribute to virus biology was tested, and this study demonstrates to the field that cellular protein chaperones may be required for maintaining the correct folding and therefore functionality of specific proteins within the virus replication complex. H uman respiratory syncytial virus (HRSV) is the leading cause of severe respiratory tract infections in newborn children worldwide (1). It infects almost all infants within the first 2 years of life and is the main cause of infant bronchiolitis. Currently, only ribav...

Murine gammaherpesvirus 68 (MHV-68) infection of laboratory mice (Mus musculus) is an established model of gammaherpesvirus pathogenesis. The fact that M. musculus is not a host in the wild prompted us to reassess MHV-68 infection in wood mice (Apodemus sylvaticus), a natural host. Here, we report significant differences in MHV-68 infection in the two species: (i) following intranasal inoculation, MHV-68 replicated in the lungs of wood mice to levels approximately 3 log units lower than in BALB/c mice; (ii) in BALB/c mice, virus replication in alveolar epithelial cells was accompanied by a diffuse, T-cell-dominated interstitial pneumonitis, whereas in wood mice it was restricted to focal granulomatous infiltrations; (iii) within wood mice, latently infected lymphocytes were abundant in inducible bronchus-associated lymphoid tissue that was not apparent in BALB/c mice; (iv) splenic latency was established in both species, but well-delineated secondary follicles with germinal centers were present in wood mice, while only poorly delineated follicles were seen in BALB/c mice; and, perhaps as a consequence, (v) production of neutralizing antibody was significantly higher in wood mice. These differences highlight the value of this animal model in the study of MHV-68 pathogenesis.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), which are aggressive malignancies associated with immunocompromised patients. For many non-viral malignancies, therapeutically targeting the ubiquitin proteasome system (UPS) has been successful. Likewise, laboratory studies have demonstrated that inhibition of the UPS might provide a promising avenue for the treatment of KSHV-associated diseases. The largest class of E3 ubiquitin ligases are the cullin-RING ligases (CRLs) that are activated by an additional ubiquitin-like protein, NEDD8. We show that pharmacological inhibition of NEDDylation (using the small molecule inhibitor MLN4924) is cytotoxic to PEL cells by inhibiting NF-κB. We also show that CRL4B is a novel regulator of latency as its inhibition reactivated lytic gene expression. Furthermore, we uncovered a requirement for NEDDylation during the reactivation of the KSHV lytic cycle. Intriguingly, inhibition prevented viral DNA replication but not lytic cycle-associated gene expression, highlighting a novel mechanism that uncouples these two features of KSHV biology. Mechanistically, we show that MLN4924 treatment precluded the recruitment of the viral pre-replication complex to the origin of lytic DNA replication (OriLyt). These new findings have revealed novel mechanisms that regulate KSHV latency and reactivation. Moreover, they demonstrate that inhibition of NEDDylation represents a novel approach for the treatment of KSHV-associated malignancies.

Overline: BIOCHEMISTRYEditor's Summary: Artificial proteins target SUMO SUMOylation is the covalent attachment of SUMO 1, SUMO 2, SUMO 3, or combinations thereof to target proteins. This posttranslational modification controls protein function and localization. Hughes et al. screened a library of artificial proteins called Affimers to identify those that bound to SUMO. By incorporating a negative selection step to remove Affimers that bound to SUMO 1, the authors identified Affimers that recognized SUMO 1, SUMO 2/ 3 (SUMO 2 and SUMO 3, which are almost identical), or all three isoforms. Biochemical and cellular assays showed that these SUMO specific Affimers (S Affs) did not interfere with SUMO conjugation or deconjugation but did inhibit a cellular stress response that required SUMO mediated protein interactions. In addition to generating S Affs that will be useful tools for studying SUMO dependent cellular processes, this study also shows the applicability of this technology for generating reagents that interfere with specific protein protein interactions, which are useful for basic research and potentially for clinical development. Furthermore, through structural analysis and molecular modelling, we explored the molecular mechanisms that may underlie their specificity in interfering with either SUMO1 mediated interactions or interactions mediated by either SUMO2 or SUMO3. Not only will these reagents enable investigation of the biological roles of SUMOylation, the Affimer technology used to generate these synthetic binding proteins could be exploited to design or validate reagents or therapeutics that target other protein protein interactions.

Two novel gammaherpesviruses were isolated, one from a field vole (Microtus agrestis) and the other from wood mice (Apodemus sylvaticus). The genome of the latter, designated wood mouse herpesvirus (WMHV), was completely sequenced. WMHV had the same genome structure and predicted gene content as murid herpesvirus 4 (MuHV4; murine gammaherpesvirus 68). Overall nucleotide sequence identity between WMHV and MuHV4 was 85 % and most of the 10 kb region at the left end of the unique region was particularly highly conserved, especially the viral tRNA-like sequences and the coding regions of genes M1 and M4. The partial sequence (71 913 bp) of another gammaherpesvirus, Brest herpesvirus (BRHV), which was isolated ostensibly from a white-toothed shrew (Crocidura russula), was also determined. The BRHV sequence was 99.2 % identical to the corresponding portion of the WMHV genome. Thus, WMHV and BRHV appeared to be strains of a new virus species. Biological characterization of WMHV indicated that it grew with similar kinetics to MuHV4 in cell culture. The pathogenesis of WMHV in wood mice was also extremely similar to that of MuHV4, except for the absence of inducible bronchus-associated lymphoid tissue at day 14 post-infection and a higher load of latently infected cells at 21 days post-infection.

HighlightsProteomics was used to identify the cellular interactome of PRRSV N protein.The interactome included translation factors and PARP-1.Inhibition of PARP-1 by the small molecule 3-AB resulted in a decrease in virus infection.Sustained treatment of PRRSV infected cells with 3-AB suggested resistance free antiviral activity.

Murine γ-herpesvirus 68 (MHV-68) infection of Mus musculus-derived strains of mice is an attractive model of γ-herpesvirus infection. Surprisingly, however, ablation of expression of MHV-68 M3, a secreted protein with broad chemokine-binding properties in vitro, has no discernable effect during experimental infection via the respiratory tract. Here we demonstrate that M3 indeed contributes significantly to MHV-68 infection, but only in the context of a natural host, the wood mouse (Apodemus sylvaticus). Specifically, M3 was essential for two features unique to the wood mouse: virus-dependent inducible bronchus-associated lymphoid tissue (iBALT) in the lung and highly organized secondary follicles in the spleen, both predominant sites of latency in these organs. Consequently, lack of M3 resulted in substantially reduced latency in the spleen and lung. In the absence of M3, splenic germinal centers appeared as previously described for MHV-68-infected laboratory strains of mice, further evidence that M3 is not fully functional in the established model host. Finally, analyses of M3's influence on chemokine and cytokine levels within the lungs of infected wood mice were consistent with the known chemokine-binding profile of M3, and revealed additional influences that provide further insight into its role in MHV-68 biology.

Establishment of persistent Epstein-Barr virus (EBV) infection requires transition from a program of full viral latency gene expression (latency III) to one that is highly restricted (latency I and 0) within memory B lymphocytes. It is well established that DNA methylation plays a critical role in EBV gene silencing, and recently the chromatin boundary protein CTCF has been implicated as a pivotal regulator of latency via its binding to several loci within the EBV genome. One notable site is upstream of the common EBNA gene promoter Cp, at which CTCF may act as an enhancer-blocking factor to initiate and maintain silencing of EBNA gene transcription. It was previously suggested that increased expression of CTCF may underlie its potential to promote restricted latency, and here we also noted elevated levels of DNA methyltransferase 1 (DNMT1) and DNMT3B associated with latency I. Within B-cell lines that maintain latency I, however, stable knockdown of CTCF, DNMT1, or DNMT3B or of DNMT1 and DNMT3B in combination did not result in activation of latency III protein expression or EBNA gene transcription, nor did knockdown of DNMTs significantly alter CpG methylation within Cp. Thus, differential expression of CTCF and DNMT1 and -3B is not critical for maintenance of restricted latency. Finally, mutant EBV lacking the Cp CTCF binding site exhibited sustained Cp activity relative to wild-type EBV in a recently developed B-cell superinfection model but ultimately was able to transition to latency I, suggesting that CTCF contributes to but is not necessarily essential for the establishment of restricted latency. E pstein-Barr virus (EBV) establishes a lifelong, largely quiescent (latent) infection within B lymphocytes of its human host.This requires the concerted actions of the viral latency-associated genes, several of which are believed to facilitate a germinal center (GC)-like reaction to promote differentiation of infected B cells into ones phenotypically defined as memory B cells and which serve as the primary reservoir of EBV within persistently infected individuals (reviewed in reference 59). During the establishment of latency in vivo, infected B cells must transition through several programs of EBV latency gene transcription, beginning with expression of the full complement of latency proteins (the latency III program), i.e., six nuclear antigens (EBNAs) and three integral plasma membrane proteins (LMPs), that is associated with a rapid EBV-induced expansion of infected cells. Thereafter, expression proceeds through a more restricted program limited to EBNA1, LMP1, and LMP2 (latency II) and ultimately to a complete restriction of EBV protein expression in the memory B cell (latency 0 [alternatively, the latency program]) (reviewed in reference 44). During subsequent periods of limited cell division, reactivation of expression of the EBV genome-maintenance protein EBNA1 alone (latency I) occurs to ensure against loss of the episomal viral genome (12).With the exception of latency 0, each of the viral latency prog...