Early Induction of Autophagy in Human Fibroblasts after Infection with Human Cytomegalovirus or Herpes Simplex Virus 1

Abstract

The infection of human fetal foreskin fibroblasts (HFFF2) with human cytomegalovirus (HCMV) resulted in the induction of autophagy. This was demonstrated by the increased lipidation of microtubule-associated protein 1 light chain 3 (LC3), a hallmark of autophagy, and by the visualization of characteristic vesicles within infected cells. The response was detected first at 2 h postinfection and persisted for at least 3 days. De novo protein synthesis was not required for the effect, since HCMV that was irradiated with UV light also elicited the response, and furthermore the continuous presence of cycloheximide did not prevent induction. Infection with herpes simplex virus type 1 (HSV-1) under conditions that inhibited viral gene expression provoked autophagy, whereas UV-irradiated respiratory syncytial virus did not. The induction of autophagy occurred when cells were infected with HCMV or HSV-1 that was gradient purified, but HCMV dense bodies and HSV-1 light particles, each of which lack nucleocapsids and genomes, were inactive. The depletion of regulatory proteins Atg5 and Atg7, which are required for autophagy, reduced LC3 modification in response to infection but did not result in any detectable difference in viral or cellular gene expression at early times after infection. The electroporation of DNA into HFFF2 cultures induced the lipidation of LC3 but double-stranded RNA did not, even though both agents stimulated an innate immune response. The results show a novel, early cellular response to the presence of the incoming virion and additionally demonstrate that autophagy can be induced by the presence of foreign DNA within cells.Autophagy is a process by which cellular organelles and abnormal proteins are degraded (recently reviewed in references 16, 43, and 75). It is an important mechanism for maintaining cell viability in times of starvation and stress, as it recycles cell components to provide nutrients. In addition, autophagy provides a means to eliminate toxic protein aggregates or damaged organelles from the cell. Three types of autophagy are recognized, named macroautophagy, microautophagy, and chaperone-mediated autophagy, and the term autophagy used here will refer only to macroautophagy. Studies of yeast first defined a number of proteins, given the prefix Atg, that control autophagy, and mammalian orthologs have been described for most of these. A simplified schematic of autophagy is shown in Fig. 1. The process is initiated by the formation of a phagophore, a double-membraned structure that can be visualized as crescent shaped by electron microscopy. The initiation of the phagophore requires the activities of two multiprotein complexes, the ULK1/2 and Beclin 1 complexes. The phagophore undergoes elongation, a stage that requires the modification of microtubule-associated protein 1 light chain 3 (LC3) by the conjugation of phosphatidylethanolamine, and the activity of a complex consisting of Atg5, Atg12, and Atg16L. The extended phagophore proceeds to engulf cell material or toxic proteins and c...

Restriction of Human Cytomegalovirus Replication by ISG15, a Host Effector Regulated by cGAS-STING Double-Stranded-DNA Sensing

“…Although ISG15 and ISG15 protein conjugates reportedly accumulated during HCMV infection (31)(32)(33), how the ISG15-encoded polypeptide itself might impact productive HCMV replication and contribute to host anti-HCMV defenses remains unknown. To better define ISG15 behavior in primary human fibroblasts (normal human dermal fibroblasts [NHDFs]) infected with HCMV, ISG15 abundance and conjugation were evaluated over a 96-h time course by immunoblotting (Fig.…”

“…In spite of the multiple mechanisms by which HCMV blunts innate immune defenses (30), the abundance of ISG15 mRNA and protein increases in response to HCMV infection (31,32). Until now, the virus-host interactions that precipitate ISG15 induction and the impact of the ISG15 polypeptide itself on productive HCMV replication remained unexplored.…”

Autophagy in cultured murine neurons infected with equid herpesvirus 1

“…Recent experimental data show that autophagy induction may occur in many ways, during different stages of infection. Some viruses can induce autophagy by the mere presence of viral DNA in a cell, independently of viral protein synthesis (McFarlane et al, 2011;Rasmussen et al, 2011). It has been demonstrated that early induction of autophagy in cells infected with HHV-1 and HHV-5 was elicited by the presence of extraneous DNA (McFarlane et al, 2011).…”

Herpes simplex virus types 1 and 2 modulate autophagy in SIRC corneal cells

“…In contrast, under conditions leading to massive cell damage, excessive autophagy possesses destructive power culminating in cell demise (Chen and Klionsky 2011). It has been shown that at the early stage of infection HSV-1 provokes autophagy (McFarlane et al 2011). However, some viral proteins (ICP34.5 and US11) synthesized during the subsequent phases of productive infection function as powerful inhibitors of the autophagic process (Tallóczy et al 2001;Tallóczy et al 2006;Orvedahl et al 2007;Lussignol et al 2013).…”

“…HSV-1 and HSV-2 are also capable of modulating autophagy, termed type II programmed cell death (Tallóczy et al 2001;Tallóczy et al 2006;McFarlane et al 2011;Lussignol et al 2013). Autophagy is an essential catabolic process that maintains cellular integrity by degrading cytoplasmic constituents and organelles (Deretic and Levine 2009).…”