Varicella-zoster virus (VZV) is a human pathogen that causes chicken pox (varicella) and shingles (zoster) (55
). Zoster is the disease associated with reactivation of latent VZV in the elderly. The virus exists as a spherical particle approximately 200 nm in diameter, including a 125-kb DNA genome enclosed in an icosahedral capsid which is itself surrounded by an amorphous shell of proteins called the tegument and an outer lipid envelope containing viral glycoproteins (9
). The most prominent viral glycoprotein is called gE and is part of the gE/gI complex (20
). Within a few days after infection, viral replication leads to the assembly of nascent viral particles in the head and neck region. A viremia ensues within T lymphocytes, after which viral particles exit the capillaries and replicate within the epidermis to cause the characteristic vesicular rash (28
). The skin vesicle is considered to be the final site of assembly and envelopment of the mature VZ virion (52
). Relatively little is known about the innate immune response within the cutaneous microenvironment (2
How a cell responds to viral infection and in turn how the virus attempts to moderate that response have been a topic of renewed research. One such response of the host cell is to increase macroautophagy (29
). Macroautophagy is a catabolic process by which whole or parts of organelles are sequestrated into double-membraned autophagosomes in the cytoplasm and then degraded when the autophagosomes fuse with lysosomes (48
). Autophagy frees amino acids and other metabolites for use in other cellular processes. Viral peptides are generated by the degradation process and become available for presentation on the cell surface via major histocompatibility complex (MHC) (11
). Thus, autophagy is thought to play a role in both innate and adaptive immunity (12
). Recent reviews summarize the range of interactions between viruses and the autophagic process (19
). Some RNA viruses, for example, hepatitis C virus, have coopted the autophagy mechanism to produce viral particles (14
). On the other hand, a complex DNA virus closely related to VZV, namely, herpes simplex virus 1 (HSV-1), actively inhibits the formation of autophagosomes via its ICP34.5 protein (34
). VZV lacks an ICP34.5-homologous protein (10
). Therefore, it was of interest when we showed that autophagy is a prominent feature in cultured cells infected with VZV, as demonstrated by immunoblotting for the LC3B marker protein as well as the autophagy adaptor p62/SQSTM1 (47
). Further, we observed numerous cytoplasmic organelles with distinctive double outer membranes characteristic of autophagosomes within electron microscopic images of the same VZV-infected cells.
Here we extend earlier observations by showing that autophagosomes are a prominent and easily detectable feature in both primary VZV infection and reactivated VZV infection in humans. Further, we show that VZV infection of cultured cells induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) to that stress. The UPR is a series of signal pathways by which a stressed cell attempts to recover homeostasis by suspending protein translation, expanding the ER, facilitating degradation processes via ER-associated degradation (ERAD), and increasing autophagy (42
). The Alwine laboratory has shown that cytomegalovirus (CMV), a betaherpesvirus, selectively induces ER stress and uses components of the UPR (particularly BiP) to form a viral assembly compartment proximal to the ER (5
). If the stressed cell fails to recover homeostasis, the UPR will initiate apoptosis.
In the VZV system, we hypothesized that overly abundant viral glycoprotein biosynthesis leads to ER stress, with increased autophagy being the result of the subsequent UPR to that stress. We tested our hypothesis by comparing autophagosome formation following VZV infection with that induced by tunicamycin, a chemical that blocks glycoprotein biosynthesis in the ER and thereby initiates ER stress. We also probed for both the alternatively spliced form of the X box protein 1 (XBP1) and the CCAAT/enhancer-binding protein homologous protein (CHOP), both transcriptional activators of the UPR in response to ER stress and therefore excellent markers for ER stress (43
). Our cumulative results suggest a role for ER stress and autophagy in VZV pathogenesis during both primary and reactivated infections. This role appears to be remarkably different from that proposed for autophagy during HSV infection (29
Autophagosomes were easily and abundantly detected in a variety of vesicle samples from human cases of varicella and zoster. Thus, we have greatly extended earlier findings in which we detected autophagosomes in a biopsy sample from one case of human zoster (47
). In other words, we have demonstrated that autophagy occurs during both primary VZV infection and VZV reactivation within the skin vesicle, the final site of virion assembly and envelopment. In turn, these results strongly suggest that autophagy is a previously unrecognized component of VZV pathogenesis. The prominence of autophagosome formation was all the more unpredictable, because the VZV genome lacks the HSV γ34.5 gene, which product can inhibit autophagy and thereby enhance viral virulence. A reasonable hypothesis, based on the HSV data with ICP34.5 null virus (30
), would have proposed that autophagy unfettered by ICP34.5 would eliminate all VZV and that disease would be minimal. The fact that VZV causes disease leads us to propose in the paragraphs below that autophagy serves a role in VZV infection entirely different from that proposed for HSV infection.
Analyses of VZV vesicle cells were important also to demonstrate that autophagy in VZV-infected cells represented an authentic environment within the human with VZV disease. In that regard, we discovered that the percentage of infected vesicular keratinocytes that contained autophagosomes was equal to or higher than that observed in VZV-infected cultured cells (>70% versus <20%). This difference is possibly due to well-known low VZV titers of virus grown in cultured cells; thus, the titer of inoculum virus is invariably low (6
). Further, the findings in vesicular keratinocytes confirmed an unexpected earlier result in VZV-infected cultured cells, namely, that autophagosome formation was detectable before VZV entered the late kinetic phase of replication. In contrast, autophagy associated with other viruses is often thought to be a relatively late event in the virus life cycle (29
). When taken together, these data suggest that autophagy may be a proviral event in that autophagy would prolong the life of a cell infected with VZV and thereby diminish apoptosis. This property would be more important for a cell-associated alphaherpesvirus, such as VZV, while less critical for the more lytic HSV-1 counterpart.
The data with tunicamycin treatment were of particular interest to our laboratory because we had used tunicamycin frequently in past experiments to determine the nonglycosylated precursor forms for the VZV glycoproteins (32
). Tunicamycin is extremely effective because the chemical blocks the enzyme GlcNAc phosphotransferase, which catalyzes the transfer of N
-acetylglucosamine-1-phosphate from UDP-N
-acetylglucosamine to dolichol phosphate, the essential first step of glycoprotein biosynthesis in the ER (51
). Thus, misfolded precursor glycoprotein forms accumulate quickly in the ER, a process now known to lead to ER stress and subsequent autophagosome accumulation. In fact, tunicamycin-treated uninfected cells are a common positive control for induction of autophagy under any other experimental condition (27
). We availed ourselves of this experimental model to show the remarkable similarity of autophagy in VZV-infected cells to autophagy in tunicamycin-treated but uninfected cells. We also repeated the tunicamycin experiment in infected cells and showed no noticeable increase in autophagosome formation. In other words, VZV infection by itself was a very effective inducer of autophagy in cultured cells.
ER stress is well recognized as one precursor to autophagy through the induction of the UPR (42
). During an earlier analysis of chromatographically purified VZV glycoprotein gE by Orbitrap mass spectrometry, we had identified four proteins associated with the UPR (including 3 heat shock proteins, HSPA5, HSPA8, and HSPD1) copurifying with gE (8
). Together with the tunicamycin data, these latter results strongly supported a role for glycoproteins in the pathway of ER stress and UPR leading to autophagy in VZV-infected cells. These results were further strengthened by our current data with transient transfections expressing VZV glycoproteins gE/gI and gH/gL. ER stress was quickly documented by increased ER size, and autophagy was demonstrated by the appearance of punctate autophagosomes within 24 h posttransfection.
One observation in this work is that the response of any given cell to a stress such as tunicamycin treatment, transient transfection of VZV glycoprotein genes, or even VZV infection is variable. For example, we documented that 30% of the cells treated with tunicamycin exhibited increased LC3B puncta typical of increased autophagosomes. While 30% is significantly more than the basal rate in untreated cells, we questioned why all of the cells which experienced the same stress did not exhibit the same response (increased autophagosomes). The answer is likely that the response of the cell to stress, such as tunicamycin treatment, may be affected by the cell cycle status of the cell. For example, a cell in a resting G0 state may not respond as quickly or as extensively as a cell in a more active metabolic state such as those associated with an actively dividing cell (e.g., G1 or G2).
As mentioned above, any condition that creates an imbalance between glycoprotein biosynthesis and the protein-folding capacity of the ER leads to ER stress, which is relieved by the UPR. In turn, the UPR activates three linked signal transduction pathways via three ER stress sensors: (i) IRE1 (inositol-requiring enzyme 1), (ii) PERK (PKR-like eIF2a kinase), and (iii) ATF6 (activating transcription factor 6). When the IRE1 pathway is activated, the endoribonuclease IRE1 executes site-specific cleavage of XBP1 mRNA to produce a spliced transcript that codes for a potent 45-kDa bZIP transcriptional activator of the UPR, called XBP1s protein. We were able to detect the XBP1s protein in VZV-infected cells by immunoblotting, further proof that ER stress had activated the UPR following VZV infection of cultured cells. To provide additional evidence for VZV-induced stress, we successfully probed for a second transcription factor called CHOP, which is closely associated with activation of the PERK UPR pathway (39
). In a similar experiment performed with antibody reagents from the same commercial source, the CHOP protein has recently been detected in cells under stress after infection with mycobacteria (44
). When taken together, our data suggest that autophagy may be a proviral event in that the UPR moderates the level of ER stress in infected cells and thus prolongs the life of an infected cell. In a recent report about murine gammaherpesvirus 68, investigators similarly proposed that herpesvirus 68 can manipulate the autophagy machinery in order to promote the survival of infected endothelial cells (46
These postulated proviral effects differ remarkably from the ascribed roles of autophagy during infection with another closely related human alphaherpesvirus, namely, HSV-1. The HSV γ34.5 neurovirulence gene interacts with Beclin 1 (Atg6 yeast homolog), an essential autophagy protein and component of a complex containing the enzymatic and regulatory subunits of class III phosphatidylinositol 3-kinase (Vps34 and Vps15) (1
). For example, recent HSV experiments suggest that the interaction of HSV ICP34.5 with Beclin 1 diminishes autophagy-mediated MHC class II antigen presentation and thereby enhances HSV-1 neuropathogenesis in an animal model (30
). Since VZV has evolved as the human herpesvirus with the smallest genome, lacking any ICP34.5 ortholog, VZV presumably has yielded its ability to circumvent the cellular autophagy pathways during its life cycle.