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

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Herpes simplex virus 1 encodes at least four genes whose functions include blocking apoptosis induced by exogenous agents (e.g., sorbitol, Fas ligand, and BAD protein) or replication-incompetent mutants (e.g., the d120 mutant lacking both copies of the ␣4 gene). U S3, one of these four genes, encodes a serine-threonine kinase that has been demonstrated to block apoptosis induced by proapoptotic cellular proteins or by the d120 mutant. The amino acid context of serine-threonine phosphorylated by U S3 is similar to that of the cAMP-dependent protein kinase PKA. We report that (i) the pattern of proteins phosphorylated by U S3 in transduced cells or in cells infected with WT virus overlaps that of phosphoproteins targeted by PKA, (ii) activation of PKA blocks apoptosis induced by d120 mutant or by BAD protein independently of U S3, (iii) US3 protein kinase phosphorylates peptides containing the serine or threonine targeted by PKA including that present in the regulatory type II␣ subunit of PKA, and (iv) in WT virus-infected cells the regulatory type II␣ subunit is phosphorylated in a US3-dependent manner. We conclude that a major determinant of the antiapoptotic activity of the U S3 protein kinase is the phosphorylation of PKA substrates by either or both enzymes.replication-defective viruses ͉ BAD protein ͉ forskolin ͉ RII␣ subunit of PKA

When grown in cultured cells, varicella-zoster virus (VZV) forms many aberrant light particles and produces low titers. Various studies have explored the reasons for such a phenotype and have pointed to impaired expression of specific late genes and at lysosomal targeting of egressing virions as possible causes. In the studies presented here, we report that the autophagic degradation pathway was induced at late time points after VZV infection of cultured cells, as documented by immunoblot analysis of the cellular proteins LC3B and p62/SQSTM1, along with electron microscopy analysis, which demonstrated the presence of both early autophagosomes and late autophagic compartments. Autophagy was induced in infected cells even in the presence of phosphonoacetic acid, an inhibitor of viral late gene expression, thus suggesting that accumulation of immediate-early and early viral gene products might be the major stimulus for its induction. We also showed that the autophagic response was not dependent on a specific cell substrate, virus strain, or type of inoculum. Finally, using immunofluorescence imaging, we demonstrated autophagosome-specific staining in human zoster vesicles but not in normal skin. Thus, our results document that this innate immune response pathway is a component of the VZV infectious cycle in both cultured cells and the human skin vesicle, the final site of virion formation in the infected human host.