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.

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IntroductionEach year, 100 million people are infected with malaria and over 1 million people die [1]. The parasite responsible for the vast majority of fatal malaria infections is Plasmodium falciparum, which can kill patients in a matter of hours.The first effective antimalarial drug was quinine, which was isolated from the bark of Cinchona. Since then, malaria has been treated with quinoline-based drugs such as chloroquine, quinine, mefloquine and primaquine, and with antifolates such as Fansidar (Sulphadoxinepyrimethamine). Unfortunately, many P. falciparum strains have now become resistant to chloroquine and some, such as those in South-East Asia, have also developed resistance to mefloquine and halofantrine [2]. Hence, with the problem of resistance on the one hand and multiple side effects on the other, it becomes inevitable to look for an alternative drug that would cure the deadly disease. Artemisinin is showing very strong potential as a non-conventional antimalarial drug. It is a sesquiterpene lactone with an endoperoxide bridge and has been isolated from Artemisia annua L., a plant belonging to the family Asteraceae. It is an annual herb native to China and known as Quinghao (A. annua) with a long history of use. This plant has now become naturalized in many countries including Argentina, Bulgaria, France, Hungary, Romania, Italy, Spain, Yugoslavia and India [3], [4], [5]. Phenotypically the plant is 50 ± 150 cm tall and may appear as small, prostrate to tall erect specimens with a woody stem. The maximum attained height is 2 m (studies at Hamdard University, New Delhi, 1993± 2000. The earliest report on the use of the extract of Quinghao (A. annua) was in the preparation of a cure for 52 kinds of ªdiseasesº that was found in the Mawanhgolui Han dynasty Tombs dating to 168 BC, which recommended its use for hemorrhoids. The use of the extract for fever including malaria was first reported in Zhon Hon Bei ji Jang AbstractArtemisinin, a sesquiterpene lactone containing an endoperoxide bridge, has been isolated from the aerial parts of Artemisia annua L. plants. It is effective against both drug-resistant and cerebral malaria-causing strains of Plasmodium falciparum. The relatively low yield (0.01 ± 0.8 %) of artemisinin in A. annua is a serious limitation to the commercialization of the drug. Therefore, the enhanced production of artemisinin either in cell/tissue culture or in the whole plant of A. annua is highly desirable. It can be achieved by a better understanding of the biochemical pathway leading to the synthesis of artemisinin and its regulation by both exogenous and endogenous factors. Furthermore, genetic engineering tools can be employed to overexpress gene(s) coding for enzyme(s) associated with the rate limiting step(s) of artemisinin biosynthesis or to inhibit the enzyme(s) of other pathway competing for its precursors. These aspects which may be employed to enhance the yield of artemisinin both in vitro and in vivo are discussed in this review.

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