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. IMPORTANCE Human 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.

Rodent herpesviruses such as murine cytomegalovirus (host, Mus musculus ), rat cytomegalovirus (host, Rattus norvegicus ), and murine gammaherpesvirus 68 (hosts, Apodemus species) are important tools for the experimental study of human herpesvirus diseases. However, alphaherpesviruses, roseoloviruses, and lymphocryptoviruses, as well as rhadinoviruses, that naturally infect Mus musculus (house mouse) and other Old World mice are unknown. To identify hitherto-unknown rodent-associated herpesviruses, we captured M. musculus , R. norvegicus , and 14 other rodent species in several locations in Germany, the United Kingdom, and Thailand. Samples of trigeminal ganglia, dorsal root ganglia, brains, spleens, and other organs, as well as blood, were analyzed with a degenerate panherpesvirus PCR targeting the DNA polymerase (DPOL) gene. Herpesvirus-positive samples were subjected to a second degenerate PCR targeting the glycoprotein B (gB) gene. The sequences located between the partial DPOL and gB sequences were amplified by long-distance PCR and sequenced, resulting in a contiguous sequence of approximately 3.5 kbp. By DPOL PCR, we detected 17 novel betaherpesviruses and 21 novel gammaherpesviruses but no alphaherpesvirus. Of these 38 novel herpesviruses, 14 were successfully analyzed by the complete bigenic approach. Most importantly, the first gammaherpesvirus of Mus musculus was discovered ( Mus musculus rhadinovirus 1 [MmusRHV1]). This virus is a member of a novel group of rodent gammaherpesviruses, which is clearly distinct from murine herpesvirus 68-like rodent gammaherpesviruses. Multigenic phylogenetic analysis, using an 8-kbp locus, revealed that MmusRHV1 diverged from the other gammaherpesviruses soon after the evolutionary separation of Epstein-Barr virus-like lymphocryptoviruses from human herpesvirus 8-like rhadinoviruses and alcelaphine herpesvirus 1-like macaviruses.

High-throughput sequencing (HTS) of virus-infected cells can be used to study in great detail the patterns of virus transcription and replication. For paramyxoviruses, and by analogy for all other negative-strand RNA viruses, we show that directional sequencing must be used to distinguish between genomic RNA and mRNA/antigenomic RNA because significant amounts of genomic RNA copurify with poly(A)-selected mRNA. We found that the best method is directional sequencing of total cell RNA, after the physical removal of rRNA (and mitochondrial RNA), because quantitative information on the abundance of both genomic RNA and mRNA/antigenomes can be simultaneously derived. Using this approach, we revealed new details of the kinetics of virus transcription and replication for parainfluenza virus (PIV) type 2, PIV3, PIV5, and mumps virus, as well as on the relative abundance of the individual viral mRNAs.

Artificial binding proteins are tools for exploring cellular processes dependent on SUMOylation.

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. Kaposi’s sarcoma-associated herpesvirus (KSHV) causes Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL), often fatal malignancies afflicting HIV-infected patients. Previous research has shown that blockade of the ubiquitin proteasome system (UPS, a normal quality control pathway that degrades cellular proteins) is able to kill KSHV-infected lymphoma cells. A large component of the UPS is made up by the protein family known as the cullin-RING ubiquitin ligases (CRLs), which are activated by NEDD8 (a process known as NEDDylation). Recently, an inhibitor of NEDDylation (MLN4924) was developed and is currently in clinical trials as an anti-cancer drug. As NEDDylation has not been investigated for many viruses, we used this to compound examine its importance in KSHV biology. Firstly we show that NEDDylation is essential for the viability of KSHV-infected lymphoma cells, and MLN4924 treatment killed these cells by blocking NF-κB activity (required for KSHV latency gene expression and KSHV-associated cancer). Furthermore, we show that NEDDylation is required for KSHV to replicate its genome, a critical step in the production of new virus particles. Therefore, this research has identified a novel molecular mechanism that governs KSHV replication. Furthermore, it demonstrates that NEDDylation is a viable target for the treatment of KSHV-associated malignancies.