抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Autophagy, or cellular self-digestion, is a cellular pathway involved in protein and organelle degradation, with an astonishing number of connections to human disease and physiology. For example, autophagic dysfunction is associated with cancer, neurodegeneration, microbial infection and ageing. Paradoxically, although autophagy is primarily a protective process for the cell, it can also play a role in cell death. Understanding autophagy may ultimately allow scientists and clinicians to harness this process for the purpose of improving human health.

/pdf/autophagy-fights-disease-through-cellular-self-digestion-5i6zc9bv8a.pdf

Autophagy fights disease through cellular self-digestion

Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. Our study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.

/pdf/ampk-and-mtor-regulate-autophagy-through-direct-14wqsz16ke.pdf

AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-28d1pi1jvo.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy

Enterovirus D68 is a re-emerging enterovirus which causes acute respiratory illness in infants. EV-D68 infection has recently been associated with Acute Flaccid Myelitis, a severe polio-like neurological disease that causes limb weakness and loss of muscle tone in infants. There is currently no FDA-approved drug or prophylactic vaccine against EV-D68. Here, we investigated the role of the histone deacetylase, SIRT-1, in autophagy and EV-D68 infection. We show that SIRT-1 plays an important role in both autophagy and EV-D68 infection. siRNA-mediated knockdown of the cellular protein blocks basal and stress-induced autophagy and reduces EV-D68 extracellular viral titers. The proviral activity of SIRT-1 does not require deacetylase activity, since transient expression of both wild-type and deacetylase-inactive SIRT-1 mutant plasmids increased EV-D68 release. In non-lytic conditions, EV-D68 is primarily released in extracellular vesicles, and SIRT-1 is required for this process. Knockdown of SIRT-1 further impedes EV-D68 release in the autophagy-deficient ATG-7 knockout cells. Knockdown of SIRT-1 also decreases titers of poliovirus (PV) and SARS-CoV-2, but not Coxsackievirus-B3 (CVB3). CVB3 is the only tested virus that fails to induce SIRT-1 translocation to the cytosol. Our data suggest a correlation between SIRT-1 translocation during viral infection and extracellular vesicle-mediated non-lytic release of infectious viral particles. SIGNIFICANCEPicornaviruses, including EV-D68, constitute a significant cause of human disease. EV-D68 infection generally causes mild respiratory tract infection in infants but has recently been implicated in a severe polio-like neurological disease, AFM. Given the lack of prophylactic vaccines or antivirals against EV-D68, identifying host factors that modulate EV-D68 infection is crucial. Here, we show that SIRT-1 regulates autophagy and EV-D68 infection. Knockdown of SIRT-1 blocked autophagy and impeded the non-lytic release of EV-D68 in extracellular vesicles. We also show that SIRT-1 modulates the release of SARS-CoV-2 and poliovirus but not Coxsackievirus-B3 virus. Our data suggest that many RNA viruses require SIRT-1 for egress and that targeting SIRT-1 could constitute a broad-spectrum antiviral strategy.

/pdf/sirt-1-connects-autophagy-and-release-of-virus-containing-1r1tgkfz.pdf

SIRT-1 connects autophagy and release of virus-containing vesicles during picornavirus infection

/pdf/isolation-of-radiolabeled-poliovirus-particles-from-h1-hela-3wiysicpxc.pdf

Isolation of Radiolabeled Poliovirus Particles from H1 HeLa Cells

Positive‐strand RNA viruses have been the cause of several recent outbreaks and epidemics, including the Zika virus epidemic in 2015, the SARS outbreak in 2003, and the ongoing SARS‐CoV‐2 pandemic. On June 18–22, 2022, researchers focusing on positive‐strand RNA viruses met for the Keystone Symposium “Positive‐Strand RNA Viruses” to share the latest research in molecular and cell biology, virology, immunology, vaccinology, and antiviral drug development. This report presents concise summaries of the scientific discussions at the symposium.

Positive‐strand RNA viruses—a Keystone Symposia report

Enterovirus D68 (EV-D68), a picornavirus traditionally associated with respiratory infections, has recently been linked to a polio-like paralytic condition known as acute flaccid myelitis (AFM). EV-D68 is understudied, and much of the field’s understanding of this virus is based on studies of poliovirus. For poliovirus, we previously showed that low pH promotes virus capsid maturation, but here we show that, for EV-D68, inhibition of compartment acidification during a specific window of infection causes a defect in capsid formation and maintenance. These phenotypes are accompanied by radical changes in the infected cell, with viral replication organelles clustering in a tight juxtanuclear grouping. Organelle acidification is critical during a narrow window from 3-4hpi, which we have termed the “transition point,” separating translation and peak RNA replication from capsid formation, maturation and egress. Our findings highlight that acidification is crucial only when vesicles convert from RNA factories to virion crucibles. Importance The respiratory picornavirus enterovirus D68 is a causative agent of Acute Flaccid Myelitis, a childhood paralysis disease identified in the last decade. Poliovirus, another picornavirus associated with paralytic disease, is a fecal-oral virus which survives acidic environments when passing from host-to-host. Here we follow up on our previous work showing a requirement for acidic intracellular compartments for maturation cleavage of poliovirus particles. Enterovirus D68 requires acidic vesicles for an earlier step, assembly and maintenance of viral particles themselves. These data have strong implications for the use of acidification blocking treatments to combat enterovirus diseases.

/pdf/enterovirus-d68-capsid-formation-and-stability-requires-2dm2ilrk.pdf

William T. Jackson

Papers

SIRT-1 connects autophagy and release of virus-containing vesicles during picornavirus infection

Isolation of Radiolabeled Poliovirus Particles from H1 HeLa Cells

Positive‐strand RNA viruses—a Keystone Symposia report

Enterovirus D68 capsid formation and stability requires acidic compartments

Transcription of the Schizosaccharomyces pombe gene cdc18+: roles of MCB elements and the DSC1 complex.