A poxvirus pseudokinase represses viral DNA replication via a pathway antagonized by its paralog kinase

Poxviruses employ sophisticated, but incompletely understood, signaling pathways that engage cellular defense mechanisms and simultaneously ensure viral factors are modulated properly. For example, the vaccinia B1 protein kinase plays a vital role in inactivating the cellular antiviral factor BAF, and likely orchestrates other pathways as well. In this study, we utilized experimental evolution of a B1 deletion virus to perform an unbiased search for suppressor mutations and identify novel pathways involving B1. After several passages of the ΔB1 virus we observed a robust increase in viral titer of the adapted virus. Interestingly, our characterization of the adapted viruses reveals that mutations correlating with a loss of function of the vaccinia B12 pseudokinase provide a striking fitness enhancement to this virus. In support of predictions that reductive evolution is a driver of poxvirus adaptation, this is clear experimental evidence that gene loss can be of significant benefit. Next, we present multiple lines of evidence demonstrating that expression of full length B12 leads to a fitness reduction in viruses with a defect in B1, but has no apparent impact on wild-type virus or other mutant poxviruses. From these data we infer that B12 possesses a potent inhibitory activity that can be masked by the presence of the B1 kinase. Further investigation of B12 attributes revealed that it primarily localizes to the nucleus, a characteristic only rarely found among poxviral proteins. Surprisingly, BAF phosphorylation is reduced under conditions in which B12 is present in infected cells without B1, indicating that B12 may function in part by enhancing antiviral activity of BAF. Together, our studies of B1 and B12 present novel evidence that a paralogous kinase-pseudokinase pair can exhibit a unique epistatic relationship in a virus, perhaps serving to enhance B1 conservation during poxvirus evolution and to orchestrate yet-to-be-discovered nuclear events during infection

The Vaccinia Virus (VACV) B1 and Cellular VRK2 Kinases Promote VACV Replication Factory Formation through Phosphorylation-Dependent Inhibition of VACV B12

Comparative examination of viral and host protein homologs reveals novel mechanisms governing downstream signaling effectors of both cellular and vi- ral origin. The vaccinia virus B1 protein kinase is involved in promoting multiple facets of the virus life cycle and is a homolog of three conserved cellular enzymes called vaccinia virus-related kinases (VRKs). Recent evidence indicates that B1 and VRK2 mediate a com- mon pathway that is largely uncharacterized but appears independent of previous VRK substrates. Interestingly, separate studies described a novel role for B1 in inhibiting vac- cinia virus protein B12, which otherwise impedes an early event in the viral lifecycle. Herein, we characterize the B1/VRK2 signaling axis to better understand their shared functions. First, we demonstrate that vaccinia virus uniquely requires VRK2 for viral repli- cation in the absence of B1, unlike other DNA viruses. Employing loss-of-function analy- sis, we demonstrate that vaccinia virus’s dependence on VRK2 is only observed in the presence of B12, suggesting that B1 and VRK2 share a pathway controlling B12. More- over, we substantiate a B1/VRK2/B12 signaling axis by examining coprecipitation of B12 by B1 and VRK2. Employing execution point analysis, we reveal that virus replication proceeds normally through early protein translation and uncoating but stalls at replica- tion factory formation in the presence of B12 activity. Finally, structure/function analyses of B1 and VRK2 demonstrate that enzymatic activity is essential for B1 or VRK2 to inhibit B12. Together, these data provide novel insights into B1/VRK signaling coregulation and support a model in which these enzymes modulate B12 in a phosphorylation-depen- dent manner

Profilin-1 regulates DNA replication forks in a context-dependent fashion by interacting with SNF2H and BOD1L

DNA replication forks are tightly controlled by a large protein network consisting of well-known core regulators and many accessory factors which remain functionally undefined. In this study, we report previously unknown nuclear functions of the actin-binding factor profilin-1 (PFN1) in DNA replication, which occur in a context-dependent fashion and require its binding to poly-L-proline (PLP)-containing proteins instead of actin. In unperturbed cells, PFN1 increases DNA replication initiation and accelerates fork progression by binding and stimulating the PLP-containing nucleosome remodeler SNF2H. Under replication stress, PFN1/SNF2H increases fork stalling and functionally collaborates with fork reversal enzymes to enable the over-resection of unprotected forks. In addition, PFN1 binds and functionally attenuates the PLP-containing fork protector BODL1 to increase the resection of a subset of stressed forks. Accordingly, raising nuclear PFN1 level decreases genome stability and cell survival during replication stress. Thus, PFN1 is a multi-functional regulator of DNA replication with exploitable anticancer potential

Inhibition of Female and Male Human Detrusor Smooth Muscle Contraction by the Rac Inhibitors EHT1864 and NSC23766

Introduction: Lower urinary tract symptoms (LUTS) due to overactive bladder (OAB) are caused by spontaneous detrusor contractions. Medical treatment with muscarinic receptor antagonists or β3-adrenoceptor agonists aims to inhibit detrusor contractions, but overall results are unsatisfactory. Consequently, improved understanding of bladder smooth muscle contraction and identification of novel compounds for its inhibition are needed to develop alternative options. A role of the GTPase Rac1 for smooth muscle contraction has been reported from the prostate, but is unknown in the human detrusor. Here, we examined effects of the Rac inhibitors NSC23766, which may also antagonize muscarinic receptors, and EHT1864 on contraction of human detrusor tissues. Methods: Female and male human detrusor tissues were obtained from radical cystectomy. Effects of NSC23766 (100 µM) and EHT1864 (100 µM) on detrusor contractions were studied in an organ bath. Results: Electric field stimulation induced frequency-dependent contractions of detrusor tissues, which were inhibited by NSC23766 and EHT1864. Carbachol induced concentration-dependent contractions. Concentration response curves for carbachol were shifted to the right by NSC23766, reflected by increased EC50 values, but unchanged Emax values. EHT1864 reduced carbachol-induced contractions, resulting in reduced Emax values for carbachol. The thromboxane analog U46619 induced concentration-dependent contractions, which remained unchanged by NSC23766, but were reduced by EHT1864. Conclusions: NSC23766 and EHT1864 inhibit female and male human detrusor contractions. NSC23766, but not EHT1864 competitively antagonizes muscarinic receptors. In addition to neurogenic and cholinergic contractions, EHT1864 inhibits thromboxane A2-induced detrusor contractions. The latter may be promising, as the origin of spontaneous detrusor contractions in OAB is noncholinergic. In vivo, both compounds may improve OAB-related LUTS

Degradation of the cancer genomic DNA deaminase APOBEC3B by SIV Vif

APOBEC3B is a newly identified source of mutation in many cancers, including breast, head/neck, lung, bladder, cervical, and ovarian. APOBEC3B is a member of the APOBEC3 family of enzymes that deaminate DNA cytosine to produce the promutagenic lesion, uracil. Several APOBEC3 family members function to restrict virus replication. For instance, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H combine to restrict HIV-1 in human lymphocytes. HIV-1 counteracts these APOBEC3s with the viral protein Vif, which targets the relevant APOBEC3s for proteasomal degradation. While APOBEC3B does not restrict HIV-1 and is not targeted by HIV-1 Vif in CD4-positive T cells, we asked whether related lentiviral Vif proteins could degrade APOBEC3B. Interestingly, several SIV Vif proteins are capable of promoting APOBEC3B degradation, with SIVmac239 Vif proving the most potent. This likely occurs through the canonical polyubiquitination mechanism as APOBEC3B protein levels are restored by MG132 treatment and by altering a conserved E3 ligase-binding motif. We further show that SIVmac239 Vif can prevent APOBEC3B mediated geno/cytotoxicity and degrade endogenous APOBEC3B in several cancer cell lines. Our data indicate that the APOBEC3B degradation potential of SIV Vif is an effective tool for neutralizing the cancer genomic DNA deaminase APOBEC3B. Further optimization of this natural APOBEC3 antagonist may benefit cancer therapy

A new role for Zinc limitation in bacterial pathogenicity: modulation of α-hemolysin from uropathogenic Escherichia coli.

Metal limitation is a common situation during infection and can have profound effects on the pathogen's success. In this report, we examine the role of zinc limitation in the expression of a virulence factor in uropathogenic Escherichia coli. The pyelonephritis isolate J96 carries two hlyCABD operons that encode the RTX toxin α-hemolysin. While the coding regions of both operons are largely conserved, the upstream sequences, including the promoters, are unrelated. We show here that the two hlyCABD operons are differently regulated. The hly II operon is efficiently silenced in the presence of zinc and highly expressed when zinc is limited. In contrast, the hly I operon does not respond to zinc limitation. Genetic studies reveal that zinc-responsive regulation of the hly II operon is controlled by the Zur metalloregulatory protein. A Zur binding site was identified in the promoter sequence of the hly II operon, and we observe direct binding of Zur to this promoter region. Moreover, we find that Zur regulation of the hly II operon modulates the ability of E. coli J96 to induce a cytotoxic response in host cell lines in culture. Our report constitutes the first description of the involvement of the zinc-sensing protein Zur in directly modulating the expression of a virulence factor in bacteria

The Vaccinia Virus (VACV) B1 and Cellular VRK2 Kinases Promote VACV Replication Factory Formation through Phosphorylation-Dependent Inhibition of VACV B12

Comparative examination of viral and host protein homologs reveals novel mechanisms governing downstream signaling effectors of both cellular and viral origin. The vaccinia virus B1 protein kinase is involved in promoting multiple facets of the virus life cycle and is a homolog of three conserved cellular enzymes called vaccinia virus-related kinases (VRKs). Recent evidence indicates that B1 and VRK2 mediate a common pathway that is largely uncharacterized but appears independent of previous VRK substrates. Interestingly, separate studies described a novel role for B1 in inhibiting vaccinia virus protein B12, which otherwise impedes an early event in the viral lifecycle. Herein, we characterize the B1/VRK2 signaling axis to better understand their shared functions. First, we demonstrate that vaccinia virus uniquely requires VRK2 for viral replication in the absence of B1, unlike other DNA viruses. Employing loss-of-function analysis, we demonstrate that vaccinia virus’s dependence on VRK2 is only observed in the presence of B12, suggesting that B1 and VRK2 share a pathway controlling B12. Moreover, we substantiate a B1/VRK2/B12 signaling axis by examining coprecipitation of B12 by B1 and VRK2. Employing execution point analysis, we reveal that virus replication proceeds normally through early protein translation and uncoating but stalls at replication factory formation in the presence of B12 activity. Finally, structure/function analyses of B1 and VRK2 demonstrate that enzymatic activity is essential for B1 or VRK2 to inhibit B12. Together, these data provide novel insights into B1/VRK signaling coregulation and support a model in which these enzymes modulate B12 in a phosphorylation-dependent manner

Efficient Gene Knockout and Genetic Interaction Screening Using the in4mer CRISPR/Cas12a Multiplex Knockout Platform

Genetic interactions mediate the emergence of phenotype from genotype, but technologies for combinatorial genetic perturbation in mammalian cells are challenging to scale. Here, we identify background-independent paralog synthetic lethals from previous CRISPR genetic interaction screens, and find that the Cas12a platform provides superior sensitivity and assay replicability. We develop the in4mer Cas12a platform that uses arrays of four independent guide RNAs targeting the same or different genes. We construct a genome-scale library, Inzolia, that is ~30% smaller than a typical CRISPR/Cas9 library while also targeting ~4000 paralog pairs. Screens in cancer cells demonstrate discrimination of core and context-dependent essential genes similar to that of CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes. Importantly, the in4mer platform offers a fivefold reduction in library size compared to other genetic interaction methods, substantially reducing the cost and effort required for these assays

High-risk human papillomavirus (HPV) screening and detection in normal, healthy patient saliva samples: a pilot cluster randomized study

Background: The human papillomaviruses (HPV) are a large family of non-enveloped DNA viruses, mainly associated with cervical cancers. Recent epidemiologic evidence has suggested that HPV may be an independent risk factor for oropharyngeal cancers. Evidence now suggests HPV may modulate the malignancy process in some tobacco- and alcohol-induced oropharynx tumors, but might also be the primary oncogenic factor for inducing carcinogenesis among some non-smokers. More evidence, however, is needed regarding oral HPV prevalence among healthy adults to estimate risk. The goal of this study was to perform an HPV screening of normal healthy adults to assess oral HPV prevalence. Methods: Healthy adult patients at a US dental school were selected to participate in this pilot study. DNA was isolated from saliva samples and screened for high-risk HPV strains HPV16 and HPV18 and further processed using qPCR for quantification and to confirm analytical sensitivity and specificity. Results: Chi-square analysis revealed the patient sample was representative of the general clinic population with respect to gender, race and age (p \u3c 0.05). Four patient samples were found to harbor HPV16 DNA, representing 2.6% of the total (n = 151). Three of the four HPV16-positive samples were from patients under 65 years of age and all four were female and Hispanic (non-White). No samples tested positive for HPV18. Conclusions: The successful recruitment and screening of healthy adult patients revealed HPV16, but not HPV18, was present in a small subset. These results provide new information about oral HPV status, which may help to contextualize results from other studies that demonstrate oral cancer rates have risen in the US among both females and minorities and in some geographic areas that are not solely explained by rates of tobacco and alcohol use. The results of this study may be of significant value to further our understanding of oral health and disease risk, as well as to help design future studies exploring the role of other factors that influence oral HPV exposure, as well as the short- and long-term consequences of oral HPV infection

Hereditary transthyretin amyloidosis: baseline characteristics of patients in the NEURO-TTR trial

Background: Hereditary transthyretin (ATTRm) amyloidosis is a rare, progressive and fatal disease with a range of clinical manifestations.Objective: This study comprehensively evaluates disease characteristics in a large, diverse cohort of patients with ATTRm amyloidosis.Methods: Adult patients (N = 172) with Stage 1 or Stage 2 ATTRm amyloidosis who had polyneuropathy were screened and enrolled across 24 investigative sites and 10 countries in the NEURO-TTR trial (www.clinicaltrials.gov, NCT01737398). Medical and disease history, quality of life, laboratory data, and clinical assessments were analyzed.Results: The NEURO-TTR patient population was diverse in age, disease severity, TTR mutation, and organ involvement. Twenty-seven different TTR mutations were present, with Val30Met being the most common (52%). One third of patients reported early onset disease (before age 50) and the average duration of neuropathy symptoms was 5.3 years. Symptoms affected multiple organs and systems, with nearly 70% of patients exhibiting broad involvement of weakness, sensory loss, and autonomic disturbance. Over 60% of patients had cardiomyopathy, with highest prevalence in the United States (72%) and lowest in South America/Australasia (33%). Cardiac biomarker NT-proBNP correlated with left ventricular wall thickness (p<.001). Quality of life, measured by Norfolk QoL-DN and SF-36 patient-reported questionnaires, was significantly impaired and correlated with disease severity.Conclusions: Baseline data from the NEURO-TTR trial demonstrates ATTRm amyloidosis as a systemic disease with deficits in multiple organs and body systems, leading to decreased quality of life. We report concomitant presentation of polyneuropathy and cardiomyopathy in most patients, and early involvement of multiple body systems