Host resistance to viral infection requires type I (α/β) and II (γ) interferon (IFN) production. Another important defense mechanism is the degradative activity of macroautophagy (herein autophagy), ...mediated by the coordinated action of evolutionarily conserved autophagy proteins (Atg). We show that the Atg5-Atg12/Atg16L1 protein complex, whose prior known function is in autophagosome formation, is required for IFNγ-mediated host defense against murine norovirus (MNV) infection. Importantly, the direct antiviral activity of IFNγ against MNV in macrophages required Atg5-Atg12, Atg7, and Atg16L1, but not induction of autophagy, the degradative activity of lysosomal proteases, fusion of autophagosomes and lysosomes, or the Atg8-processing protein Atg4B. IFNγ, via Atg5-Atg12/Atg16L1, inhibited formation of the membranous cytoplasmic MNV replication complex, where Atg16L1 localized. Thus, the Atg5-Atg12/Atg16L1 complex performs a pivotal, nondegradative role in IFNγ-mediated antiviral defense, establishing that multicellular organisms have evolved to use portions of the autophagy pathway machinery in a cassette-like fashion for host defense.
Display omitted
► Atg5-Atg12/Atg16L1 and Atg7 are required for IFNγ to inhibit norovirus replication ► The degradative activity of autophagy is not required for the antiviral activity of IFNγ ► IFNγ, via autophagy proteins, inhibits the formation of the replication complex
Variation in the presentation of hereditary immunodeficiencies may be explained by genetic or environmental factors. Patients with mutations in HOIL1 (RBCK1) present with amylopectinosis-associated ...myopathy with or without hyper-inflammation and immunodeficiency. We report that barrier-raised HOIL-1-deficient mice exhibit amylopectin-like deposits in the myocardium but show minimal signs of hyper-inflammation. However, they show immunodeficiency upon acute infection with Listeria monocytogenes, Toxoplasma gondii or Citrobacter rodentium. Increased susceptibility to Listeria was due to HOIL-1 function in hematopoietic cells and macrophages in production of protective cytokines. In contrast, HOIL-1-deficient mice showed enhanced control of chronic Mycobacterium tuberculosis or murine γ-herpesvirus 68 (MHV68), and these infections conferred a hyper-inflammatory phenotype. Surprisingly, chronic infection with MHV68 complemented the immunodeficiency of HOIL-1, IL-6, Caspase-1 and Caspase-1;Caspase-11-deficient mice following Listeria infection. Thus chronic herpesvirus infection generates signs of auto-inflammation and complements genetic immunodeficiency in mutant mice, highlighting the importance of accounting for the virome in genotype-phenotype studies.
Pervasive transcription is observed in a wide range of organisms, including humans, mice, and viruses, but the functional significance of the resulting transcripts remains uncertain. Current genetic ...approaches are often limited by their emphasis on protein-coding open reading frames (ORFs). We previously identified extensive pervasive transcription from the murine gammaherpesvirus 68 (MHV68) genome outside known ORFs and antisense to known genes (termed expressed genomic regions EGRs). Similar antisense transcripts have been identified in many other herpesviruses, including Kaposi's sarcoma-associated herpesvirus and human and murine cytomegalovirus. Despite their prevalence, whether these RNAs have any functional importance in the viral life cycle is unknown, and one interpretation is that these are merely "noise" generated by functionally unimportant transcriptional events. To determine whether pervasive transcription of a herpesvirus genome generates RNA molecules that are functionally important, we used a strand-specific functional approach to target transcripts from thirteen EGRs in MHV68. We found that targeting transcripts from six EGRs reduced viral protein expression, proving that pervasive transcription can generate functionally important RNAs. We characterized transcripts emanating from EGRs 26 and 27 in detail using several methods, including RNA sequencing, and identified several novel polyadenylated transcripts that were enriched in the nuclei of infected cells. These data provide the first evidence of the functional importance of regions of pervasive transcription emanating from MHV68 EGRs. Therefore, studies utilizing mutation of a herpesvirus genome must account for possible effects on RNAs generated by pervasive transcription. IMPORTANCE The fact that pervasive transcription produces functionally important RNAs has profound implications for design and interpretation of genetic studies in herpesviruses, since such studies often involve mutating both strands of the genome. This is a common potential problem; for example, a conservative estimate is that there are an additional 73,000 nucleotides transcribed antisense to annotated ORFs from the 119,450-bp MHV68 genome. Recognizing the importance of considering the function of each strand of the viral genome independently, we used strand-specific approaches to identify six regions of the genome encoding transcripts that promoted viral protein expression. For two of these regions, we mapped novel transcripts and determined that targeting transcripts from these regions reduced viral replication and the expression of other viral genes. This is the first description of a function for these RNAs and suggests that novel transcripts emanating from regions of pervasive transcription are critical for the viral life cycle.
Pathogenic simian immunodeficiency virus (SIV) infection is associated with enteropathy, which likely contributes to AIDS progression. To identify candidate etiologies for AIDS enteropathy, we used ...next-generation sequencing to define the enteric virome during SIV infection in nonhuman primates. Pathogenic, but not nonpathogenic, SIV infection was associated with significant expansion of the enteric virome. We identified at least 32 previously undescribed enteric viruses during pathogenic SIV infection and confirmed their presence by using viral culture and PCR testing. We detected unsuspected mucosal adenovirus infection associated with enteritis as well as parvovirus viremia in animals with advanced AIDS, indicating the pathogenic potential of SIV-associated expansion of the enteric virome. No association between pathogenic SIV infection and the family-level taxonomy of enteric bacteria was detected. Thus, enteric viral infections may contribute to AIDS enteropathy and disease progression. These findings underline the importance of metagenomic analysis of the virome for understanding AIDS pathogenesis.
Display omitted
► The enteric virome expands during pathogenic, but not nonpathogenic, SIV infection ► Pathogenic SIV-infected rhesus monkeys shed multiple enteric viruses ► Viruses are associated with intestinal pathology during SIV infection ► Metagenomic analysis of the enteric virome is important to study AIDS pathogenesis
Monkeys infected with pathogenic SIV manifest an expanded enteric virome that includes examples of previously uncharacterized viruses. Viruses within the cohort are associated with intestinal damage, suggesting a link to the intestinal pathologies that develop in HIV-infected patients.
Abstract 1137
Platelets contain in their alpha granules ∼2.5% of the protein S in blood. It has been suggested that this protein S supports the anticoagulant activity of exogenous activated protein C ...(APC), but it is not known whether protein S that is released from stimulated platelets can exert anticoagulant activity that is independent of APC and TFPI. We recently showed that at least some of the anticoagulant activity of plasma protein S is independent of APC and TFPI, although data suggested that plasma protein S may also have TFPI-dependent activity.
To determine if platelet protein S has anticoagulant activity that is independent of APC and TFPI, prothrombinase and extrinsic FXase reactions were initiated on the surface of fresh stimulated or unstimulated washed platelets in the presence and absence of blocking antibodies against APC, TFPI, and/or protein S, or in the presence and absence of purified plasma-derived protein S. Platelets were adjusted to a concentration of 0.7 to 2 × 10e8/ml, which contained 2.3–6.5 nM total platelet protein S. The last platelet wash contained negligible amounts of plasma protein S.
Neutralizing anti-protein S antibodies allowed up to 5.7-fold (mean: 2.1 ± 1.5 –fold, n=13) more thrombin generation on calcium ionophore-stimulated platelets following supplementation with 50–500 pM FXa and 600 nM prothrombin, and allowed up to 2.5-fold (mean: 1.7 ± 0.7 –fold, n=11) more thrombin generation on platelets that were not ionophore-stimulated but were gradually stimulated following FXa and prothrombin supplementation. Anti-protein S antibodies had no effect on thrombin generation on platelets that were treated with prostaglandin E1 (PGE1) to suppress platelet activation and then supplemented with procoagulants. This implies that platelet protein S is released from stimulated platelets and downregulates thrombin generation on platelets, and that neutralizing anti-protein S antibodies block this activity of protein S. Anti-protein S antibodies allowed up to 1.8-fold (mean 1.5 ± 0.2 –fold, n=8) more FXa generation on the surface of stimulated platelets supplemented with 5 pM TF, 100 pM FVIIa, and 160 nM FX, but anti-protein S antibodies had no effect on FXa generation on platelets treated with PGE1. Most of these experiments were performed in the presence of neutralizing antibodies against TFPI and APC, but thrombin and FXa generation on platelets under the varying conditions described were unaffected by the presence of these neutralizing antibodies. Purified plasma-derived zinc-containing protein S downregulated thrombin and FXa generation on platelets (IC50 = 6–18 nM PS) and in plasma >10-fold more potently than zinc-deficient protein S. We could not demonstrate a synergistic anticoagulant effect when TFPI was combined with zinc-deficient protein S in the presence of stimulated platelets and procoagulant proteins.
Protein S that is released from stimulated platelets exerts anticoagulant activity that is independent of TFPI and APC.
No relevant conflicts of interest to declare.
Anticoagulant plasma protein S (PS) is essential for maintaining haemostatic balance. About 2.5% of PS is stored in platelets and released upon platelet stimulation. So far, little is known about the ...functionality and importance of platelet (plt)PS. A platelet-associated protease cleaves plasma-derived (pd)PS and pltPS in the “thrombin-sensitive region”, abolishing activated protein C (APC) cofactor activity. However, we showed that cleaved PS retains APC-independent anticoagulant activities (“PS-direct”). To investigate whether pltPS or pdPS exert PS-direct on platelets or platelet-shed microparticles, thrombin and factor (F)Xa generation on unstimulated or stimulated washed platelets and microparticles were measured. Western blotting revealed that pltPS and pdPS bound to washed, stimulated platelets and microparticles, and that pltPS had slower electrophoretic mobility than pdPS. Platelet stimulation in the presence of inhibitory anti-PS antibodies resulted in 2.6 ± 1.6-fold (p
The objective of the study was to assess the impact of multifaceted clinical pharmacist-led antimicrobial stewardship (AMS) program on the rational use of antibiotics for patients who receive ...vascular and interventional radiology therapies.
A quasi-experimental retrospective intervention design with a comparison group was applied to the practice of antibiotic use in the department of vascular and interventional radiology in a Chinese tertiary hospital. We used difference-in-differences (DID) analysis to compare outcomes before and after the AMS intervention between the intervention group and control group, to determine whether intervention would lead to changes in irrationality of antibiotic prescribing, antibiotic utilization, cost of antibiotics, and length of hospital stay.
The DID results showed that the intervention group was associated with a reduction in the average consumption of antibiotics (
= 0.017) and cost of antibiotics (
= 0.006) and cost per defined daily dose (DDD) (
= 0.000). There were no significant differences in the mean change of total costs and length of stay between the two groups (
> 0.05). The average inappropriate score of perioperative antimicrobial prophylaxis in the intervention group declined by 0.23, while it decreased by 0.02 in the control group 0.21 (95% CI, -0.271 to -0.143);
= 0.000. The average inappropriate score of non-surgical antimicrobial prophylaxis in the intervention group declined by 0.14, while it increased by 0.02 in the control group 0.16 (95% CI, -0.288 to -0.035);
= 0.010. The average inappropriate score of the therapeutic use of antibiotics in the intervention group declined by 0.07, while it decreased by 0.01 in the control group 0.06 (95% CI, -0.115 to -0.022);
= 0.003.
This study provides evidence that implementation of AMS interventions was associated with a marked reduction of antibiotic use, cost of antibiotics, and irrationality of antibiotic prescribing in China.
PDF
