Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome

Proteasomes are central regulators of protein homeostasis in eukaryotes. Proteasome function is vulnerable to environmental insults, cellular protein imbalance and targeted pharmaceuticals. Yet, mechanisms that cells deploy to counteract inhibition of this central regulator are little understood. To find such mechanisms, we reduced flux through the proteasome to the point of toxicity with specific inhibitors and performed genome-wide screens for mutations that allowed cells to survive. Counter to expectation, reducing expression of individual subunits of the proteasome's 19S regulatory complex increased survival. Strong 19S reduction was cytotoxic but modest reduction protected cells from inhibitors. Protection was accompanied by an increased ratio of 20S to 26S proteasomes, preservation of protein degradation capacity and reduced proteotoxic stress. While compromise of 19S function can have a fitness cost under basal conditions, it provided a powerful survival advantage when proteasome function was impaired. This means of rebalancing proteostasis is conserved from yeast to humans

CRISPR/Cas9, a powerful tool to target human herpesviruses

Over 90% of the adult population is infected with one or multiple herpesviruses. These viruses are characterized by their ability to establish latency, where the host is unable to clear the invader from infected cells resulting in a lifelong infection. Herpesviruses cause a wide variety of (recurrent) diseases such as cold sores, shingles, congenital defects and several malignancies. Although the productive phase of a herpesvirus infection can often be efficiently limited by nucleoside analogs, these drugs are ineffective during a latent herpesvirus infection and are therefore unable to clear herpesviruses from the human host. Advances in genome engineering using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 facilitates virus research and may hold potential to treat or cure previously incurable herpesvirus infections by directly targeting these viruses within infected cells. Here, we review recent applications of the CRISPR/Cas9 system for herpesviral research and discuss the therapeutic potential of the system to treat, or even cure, productive and latent herpesviral infections

Viral gene delivery vectors: the next generation medicines for immune-related diseases

Viruses have evolved to efficiently express their genes in host cells, which makes them ideally suited as gene delivery vectors for gene and immunotherapies. Replication competent (RC) viral vectors encoding foreign or self-proteins induce strong T-cell responses that can be used for the development of effective cancer treatments. Replication-defective (RD) viral vectors encoding self-proteins are non-immunogenic when introduced in a host naïve for the cognate virus. RD viral vectors can be used to develop gene replacement therapies for genetic disorders and tolerization therapies for autoimmune diseases and allergies. Degenerative/inflammatory diseases are associated with chronic inflammation and immune responses that damage the tissues involved. These diseases therefore strongly resemble autoimmune diseases. This review deals with the use of RC and RD viral vectors for unraveling the pathogenesis of immune-related diseases and their application to the development of the next generation prophylactics and therapeutics for todays’ major diseases

BUB1 Is Essential for the Viability of Human Cells in which the Spindle Assembly Checkpoint Is Compromised

Summary: The spindle assembly checkpoint (SAC) ensures faithful segregation of chromosomes. Although most mammalian cell types depend on the SAC for viability, we found that human HAP1 cells can grow SAC independently. We generated MAD1- and MAD2-deficient cells and mutagenized them to identify synthetic lethal interactions, revealing that chromosome congression factors become essential upon SAC deficiency. Besides expected hits, we also found that BUB1 becomes essential in SAC-deficient cells. We found that the BUB1 C terminus regulates alignment as well as recruitment of CENPF. Second, we found that BUBR1 was not essential in SAC-deficient HAP1 cells. We confirmed that BUBR1 does not regulate chromosome alignment in HAP1 cells and that BUB1 does not regulate chromosome alignment through BUBR1. Taken together, our data resolve some long-standing questions about the interplay between BUB1 and BUBR1 and their respective roles in the SAC and chromosome alignment

CRISPR/Cas9-Mediated Genome Editing of Herpesviruses Limits Productive and Latent Infections

Herpesviruses infect the majority of the human population and can cause significant morbidity and mortality. Herpes simplex virus (HSV) type 1 causes cold sores and herpes simplex keratitis, whereas HSV-2 is responsible for genital herpes. Human cytomegalovirus (HCMV) is the most common viral cause of congenital defects and is responsible for serious disease in immuno-compromised individuals. Epstein-Barr virus (EBV) is associated with infectious mononucleosis and a broad range of malignancies, including Burkitt’s lymphoma, nasopharyngeal carcinoma, Hodgkin’s disease, and post-transplant lymphomas. Herpesviruses persist in their host for life by establishing a latent infection that is interrupted by periodic reactivation events during which replication occurs. Current antiviral drug treatments target the clinical manifestations of this productive stage, but they are ineffective at eliminating these viruses from the infected host. Here, we set out to combat both productive and latent herpesvirus infections by exploiting the CRISPR/Cas9 system to target viral genetic elements important for virus fitness. We show effective abrogation of HCMV and HSV-1 replication by targeting gRNAs to essential viral genes. Simultaneous targeting of HSV-1 with multiple gRNAs completely abolished the production of infectious particles from human cells. Using the same approach, EBV can be almost completely cleared from latently infected EBV-transformed human tumor cells. Our studies indicate that the CRISPR/Cas9 system can be effectively targeted to herpesvirus genomes as a potent prophylactic and therapeutic anti-viral strategy that may be used to impair viral replication and clear latent virus infection

Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome

Proteasomes are central regulators of protein homeostasis in eukaryotes. Proteasome function is vulnerable to environmental insults, cellular protein imbalance and targeted pharmaceuticals. Yet, mechanisms that cells deploy to counteract inhibition of this central regulator are little understood. To find such mechanisms, we reduced flux through the proteasome to the point of toxicity with specific inhibitors and performed genome-wide screens for mutations that allowed cells to survive. Counter to expectation, reducing expression of individual subunits of the proteasome's 19S regulatory complex increased survival. Strong 19S reduction was cytotoxic but modest reduction protected cells from inhibitors. Protection was accompanied by an increased ratio of 20S to 26S proteasomes, preservation of protein degradation capacity and reduced proteotoxic stress. While compromise of 19S function can have a fitness cost under basal conditions, it provided a powerful survival advantage when proteasome function was impaired. This means of rebalancing proteostasis is conserved from yeast to humans. DOI: http://dx.doi.org/10.7554/eLife.08467.00

Anti-HSV-1 CRISPR gRNAs are ineffective in targeting quiescent HSV-1 but do abrogate replication of reactivated HSV-1.

<p><b>a)</b> MRC5 cells exogenously expressing Cas9 were infected with quiescent HSV-1-eGFP. Anti-HSV-1 gRNAs were introduced into the MRC5-Cas9 cells via lentiviral transduction and cells were subsequently superinfected with HCMV to reactivate latent HSV-1. The percentage of cells with replicating HSV-1 were assessed 3 days post HCMV superinfection by flow cytometry. Two quiescent control (‘cells alone’) samples are presented: one not superinfected with HCMV to assess spontaneous HSV-1 reactivation levels, and one superinfected with HCMV to assess the reactivation potential and subsequent HSV-1 replication in these cells. Control vector corresponds to empty gRNA-vector. <b>b)</b> Relative amount of HSV-1 genomes in quiescent HSV-1-eGFP cells transduced with the indicated gRNAs as assessed by TaqMan qPCR. DNA input was normalized to RNAseP levels. The relative HSV-1 content was compared to quiescent MRC5 cells transduced with empty vector control lentivirus. <b>c)</b> Analysis of anti-HSV-1 genome editing by next generation sequencing. The percentage of WT sequences at the indicated target sites is presented upon introduction of the corresponding gRNA. For UL8 and UL52, two samples were analyzed. In control vector treated quiescent HSV-1 cells, no CRISPR/Cas9 editing was observed at these target sites (gRNA specific indels <0.1%). CRISPR/Cas9 genome editing was only observed for UL52 #2 and UL8 #2 where CRISPR/Cas9 editing was apparent in ±6 and ±1% of sequences. The nature of these mutations is presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005701#ppat.1005701.s004" target="_blank">S4 Fig</a>.</p

Editing of the EBV genome in latently infected tumor cells using CRISPR/Cas9.

<p><b>a)</b> Latently infected gastric carcinoma SNU719 cells were transduced with lentiviral CRISPR/Cas vectors targeting the indicated EBV miRNA genes. The lines were subsequently selected with puromycin for 2 days and allowed to recover for 12 days. The activity of the targeted miRNAs was subsequently monitored by introduction of the indicated miRNA sensor vectors and assessment of the mCherry reporter expression after 4 days. Increased sensor expression indicates a loss of EBV miRNAs. The activities for the EBV miRNAs in the absence of gRNAs is presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005701#ppat.1005701.s001" target="_blank">S1 Fig</a>. <b>b)</b> Sequencing of CRISPR-targeted EBV genomes indicates editing at the target sites. The EBV genomic locus of BART5 and BART16 were amplified by PCR, cloned in a DNA cloning vector, and subjected to Sanger DNA sequencing. The miRNA sequence is presented in yellow, the gRNA-target sites are displayed in bold, the PAM sequence as red, underlined text, and the cleavage site as a triangle. The number of times each variant has been sequenced is indicated.</p

Simultaneous targeting of HSV-1 with two gRNAs completely impairs virus replication.

<p><b>a)</b> Vero cells were transduced with the indicated single or double gRNAs and subsequently infected with HSV-1-eGFP at an MOI of 0.5. Cells were analyzed for eGFP-expression by flow cytometry at 1, 2, and 3 dpi to assess the percentage of virus-infected cells. <b>b)</b> Similar experiment as in a) but now performed in human MRC5 cells and at an MOI of 0.005, as MRC5 cells are more susceptible to HSV-1 infection. Cells were analyzed for eGFP-expression by flow cytometry at 1 dpi and 3 dpi to assess the percentage of successfully infected cells. <b>c)</b> Supernatants from b) were subjected to plaque assays to quantify the infectious HSV-1 titer produced by gRNA-expressing MRC5 cells that had been infected with HSV-1-eGFP three days earlier. Plaques were scored if visible by eye. <b>d)</b> Plaques from c) obtained after infection of cells with HSV-1 harvested from control or anti-<i>UL8</i> gRNA-expressing MRC5-cells were analyzed by light-microscopy. Whereas large plaques (‘P’) are observed in infected cells not carrying any gRNAs, virus harvested from UL8 gRNA- expressing cells induced microplaques. In double gRNA-treated cells, no signs of infection were observed. For all bar diagrams, measurements of triplicate experiments are presented + STD.</p

Anti-HCMV gRNAs efficiently abrogate HCMV replication in human cells.

<p><b>a)</b> Targeting essential HCMV genes with CRISPR/Cas9 impairs HCMV replication. MRC5 cells transduced with the indicated gRNAs were infected with HCMV-eGFP strain TB40/E at an MOI of 0.05 and subjected to flow cytometry at 2, 5, 8, and 11 dpi to assess the percentage of eGFP-positive infected cells. For each essential gene, four different gRNAs were monitored. Besides targeting human genes as controls, gRNAs targeting nonessential HCMV genes US6, US7, and US11 were included. <b>b)</b> Anti-HCMV gRNAs impair replication of both TB40/E and AD169 strains with the exception of anti-UL84 gRNAs. gRNA-expressing MRC5 cells were infected with eGFP-tagged TB40/E or AD169 and the percentage of eGFP-expressing cells was monitored at 2, 5, and 8 dpi. For bar diagrams in a) triplicate measurements + STD are presented. Bar diagrams in b) are from (at least) duplicate measurements.</p