Endoplasmic Reticulum Chaperones Are Involved in the Morphogenesis of Rotavirus Infectious Particles

We have previously shown that multifunctional calreticulin (CRT), which resides in the endoplasmic reticulum (ER) and is involved in ER-associated protein processing, responds to infection with white spot syndrome virus (WSSV) by increasing mRNA and protein expression and by forming a complex with gC1qR and thereby delaying apoptosis. Here, we show that CRT can directly interact with WSSV structural proteins, including VP15 and VP28, during an early stage of virus infection. The binding of VP28 with CRT does not promote WSSV entry, and CRT-VP15 interaction was detected in the viral genome in virally infected host cells and thus may have an effect on WSSV replication. Moreover, CRT was detected in the viral envelope of purified WSSV virions. CRT was also found to be of high importance for proper oligomerization of the viral structural proteins VP26 and VP28, and when CRT glycosylation was blocked with tunicamycin, a significant decrease in both viral replication and assembly was detected. Together, these findings suggest that CRT confers several advantages to WSSV, from the initial steps of WSSV infection to the assembly of virions. Therefore, CRT is required as a "vital factor" and is hijacked by WSSV for its replication cycle. IMPORTANCEWhite spot syndrome virus (WSSV) is a double-stranded DNA virus and the cause of a serious disease in a wide range of crustaceans that often leads to high mortality rates. We have previously shown that the protein calreticulin (CRT), which resides in the endoplasmic reticulum (ER) of the cell, is important in the host response to the virus. In this report, we show that the virus uses this host protein to enter the cell and to make the host produce new viral structural proteins. Through its interaction with two viral proteins, the virus "hijacks" host calreticulin and uses it for its own needs. These findings provide new insight into the interaction between a large DNA virus and the host protein CRT and may help in understanding the viral infection process in general.

Section: Discussionsupporting

confidence: 78%

mentioning

confidence: 99%

Hijacking of Host Calreticulin Is Required for the White Spot Syndrome Virus Replication Cycle

Watthanasurorot

Guo

Tharntada

et al. 2014

“…Further experiments are needed to determine if this role for GRP78 is specific for particular DENV proteins or is a more generalized requirement for DENV polyprotein production. This role for GRP78 in chaperoning DENV protein production contrasts with the lack of a requirement for HMCV protein production for GRP78 (3,27). Knockdown of GRP78 in HCMV infection leads to intracellular viral-particle accumulation, and thus, HCMV needs GRP78 to chaperone virion particle egress but not HCMV protein production itself.…”

Section: Discussionmentioning

confidence: 67%

Dengue Virus Infection Induces Upregulation of GRP78, Which Acts To Chaperone Viral Antigen Production

Wati

Soo

Zilm

et al. 2009

Dengue virus (DENV) pathogenesis is related to the host responses to viral infection within target cells, and therefore, this study assessed intracellular changes in host proteins following DENV infection. Two-dimensional gel electrophoresis and mass spectrometry identified upregulation of the host endoplasmic reticulum (ER) chaperone GRP78 in K562 cells following DENV infection, in the absence of virus-induced cell death. Upregulation of GRP78 in DENV-infected cells was confirmed by immunostaining and confocal microscopy and by Western blot analysis and was also observed in DENV-infected primary monocyte-derived macrophages, a natural target cell type for DENV infection. GRP78 was upregulated in both DENV antigen-positive and -negative cells in the DENV-infected culture, suggesting a bystander effect, with the highest GRP78 levels coincident with high-level DENV antigen production and infectious-virus release. Transfection of target cells to express GRP78 prior to DENV challenge did not affect subsequent DENV infection, but cleavage of GRP78 with the SubAB toxin, during an established DENV infection, yielded a 10-to 100-fold decrease in infectiousvirus release, loss of intracellular DENV particles, and a dramatic decrease in intracellular DENV antigen. However, DENV RNA levels were unchanged, indicating normal DENV RNA replication but altered DENV antigen levels in the absence of GRP78. Thus, GRP78 is upregulated by DENV infection and is necessary for DENV antigen production and/or accumulation. This may be a common requirement for viruses such as flaviviruses that depend heavily on the ER for coordinated protein production and processing.The pathogenesis of dengue virus (DENV) disease is multifactorial, and many of the clinical manifestations of the disease, including the life-threatening DENV-induced hemorrhage, may be mediated by the host responses to infection. Many studies have defined altered host responses during DENV infection, including activation of T cells (19) and altered levels of circulating factors in patients (13) and altered release of cytokines and chemokines from DENV-infected cells (8). Transcriptome analyses of DENV-infected endothelial cells (23, 45), HepG2 cells (9), circulating patient cells (39), or peripheral blood mononuclear cells from DENV-infected macaque monkeys (37) have identified alterations in transcripts involved in a variety of cellular processes, including the innate immune response, cell signaling, and metabolic processes. A recent study examined proteomic changes in DENVinfected HepG2 cells and identified 17 altered cellular proteins, including 2 proteins for which the changes were confirmed (32). In the current study, we performed a proteomic analysis of changes due to DENV infection in a target cell population that is relevant to a natural DENV infection and in which DENV-induced cytopathic effect (CPE) and the cellular death response are absent. Two-dimensional gel electrophoresis (2DGE) identified upregulation of glucose-regulated protein 78 (GRP78), otherwise known a...

“…RNA interference studies have demonstrated an essential role of GRP78 in maturation and egress of rotavirus and DENV (60,61). GRP78 was also identified as a protein that was secreted from JEV-infected BHK-21 cells and was associated with JEV virions (62).…”

Section: Discussionmentioning

confidence: 99%

GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells

Nain

Mukherjee

Karmakar

et al. 2017

126

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the leading cause of viral encephalitis in Southeast Asia with potential to become a global pathogen. Here, we identify glucose-regulated protein 78 (GRP78) as an important host protein for virus entry and replication. Using the plasma membrane fractions from mouse neuronal (Neuro2a) cells, mass spectroscopy analysis identified GRP78 as a protein interacting with recombinant JEV envelope protein domain III. GRP78 was found to be expressed on the plasma membranes of Neuro2a cells, mouse primary neurons, and human epithelial Huh-7 cells. Antibodies against GRP78 significantly inhibited JEV entry in all three cell types, suggesting an important role of the protein in virus entry. Depletion of GRP78 by small interfering RNA (siRNA) significantly blocked JEV entry into Neuro2a cells, further supporting its role in virus uptake. Immunofluorescence studies showed extensive colocalization of GRP78 with JEV envelope protein in virus-infected cells. This interaction was also confirmed by immunoprecipitation studies. Additionally, GRP78 was shown to have an important role in JEV replication, as treatment of cells post-virus entry with subtilase cytotoxin that specifically cleaved GRP78 led to a substantial reduction in viral RNA replication and protein synthesis, resulting in significantly reduced extracellular virus titers. Our results indicate that GRP78, an endoplasmic reticulum chaperon of the HSP70 family, is a novel host factor involved at multiple steps of the JEV life cycle and could be a potential therapeutic target.IMPORTANCE Recent years have seen a rapid spread of mosquito-borne diseases caused by flaviviruses. The flavivirus family includes West Nile, dengue, Japanese encephalitis, and Zika viruses, which are major threats to public health with potential to become global pathogens. JEV is the major cause of viral encephalitis in several parts of Southeast Asia, affecting a predominantly pediatric population with a high mortality rate. This study is focused on identification of crucial host factors that could be targeted to cripple virus infection and ultimately lead to development of effective antivirals. We have identified a cellular protein, GRP78, that plays a dual role in virus entry and virus replication, two crucial steps of the virus life cycle, and thus is a novel host factor that could be a potential therapeutic target.