Vesicular Stomatitis Virus-Based Epstein-Barr Virus Vaccines Elicit Strong Protective Immune Responses.

Epstein-Barr virus (EBV), the first identified human tumor virus, is etiologically associated with various kinds of malignant and benign diseases, accounting for 265,000 cancer incident cases and 164,000 cancer deaths in 2017. EBV prophylactic vaccine development has been gp350 centered for several decades. However, clinical studies show that gp350-centered vaccines fail to prevent EBV infection. Advances in the EBV infection mechanisms shed light on gB and gHgL, the two key components of the infection apparatus. In this study, for the first time, we utilized recombinant vesicular stomatitis virus (VSV) to display EBV gB (VSV-ΔG-gB/gB-G) or gHgL (VSV-ΔG-gHgL). In vitro studies confirmed successful virion production and glycoprotein presentation on the virion surface. In mouse models, VSV-ΔG-gB/gB-G or VSV-ΔG-gHgL elicited potent humoral responses. Neutralizing antibodies elicited by VSV-ΔG-gB/gB-G were prone to prevent B cell infection, while those elicited by VSV-ΔG-gHgL were prone to prevent epithelial cell infection. Combinatorial vaccination yields an additive effect. The ratio of endpoint neutralizing antibody titers to the endpoint total IgG titers immunized with VSV-ΔG-gHgL was approximately 1. The ratio of IgG1/IgG2a after VSV-ΔG-gB/gB-G immunization was approximately 1 in a dose-dependent, adjuvant-independent manner. Taken together, VSV-based EBV vaccines can elicit a high ratio of epithelial and B lymphocyte neutralizing antibodies, implying their unique potential as EBV prophylactic vaccine candidates. IMPORTANCE Epstein-Barr virus (EBV), one of the most common human viruses and the first identified human oncogenic virus, accounted for 265,000 cancer incident cases and 164,000 cancer deaths in 2017 as well as millions of nonmalignant disease cases. So far, no prophylactic vaccine is available to prevent EBV infection. In this study, for the first time, we reported the VSV-based EBV vaccines presenting two key components of the EBV infection apparatus, gB and gHgL. We confirmed potent antigen-specific antibody generation; these antibodies prevented EBV from infecting epithelial cells and B cells, and the IgG1/IgG2a ratio indicated balanced humoral-cellular responses. Taken together, we suggest VSV-based EBV vaccines are potent prophylactic candidates for clinical studies and help eradicate numerous EBV-associated malignant and benign diseases.

Inclusion bodies of human parainfluenza virus type 3 inhibit antiviral stress granule formation by shielding viral RNAs.

Viral invasion triggers the activation of the host antiviral response. Besides the innate immune response, stress granules (SGs) also act as an additional defense response to combat viral replication. However, many viruses have evolved various strategies to suppress SG formation to facilitate their own replication. Here, we show that viral mRNAs derived from human parainfluenza virus type 3 (HPIV3) infection induce SG formation in an eIF2α phosphorylation- and PKR-dependent manner in which viral mRNAs are sequestered and viral replication is inhibited independent of the interferon signaling pathway. Furthermore, we found that inclusion body (IB) formation by the interaction of the nucleoprotein (N) and phosphoprotein (P) of HPIV3 correlated with SG suppression. In addition, co-expression of P with NL478A (a point mutant of N, which is unable to form IBs with P) or with NΔN10 (lacking N-terminal 10 amino acids of N, which could form IBs with P but was unable to synthesize or shield viral RNAs) failed to inhibit SG formation, suggesting that inhibition of SG formation also correlates with the capacity of IBs to synthesize and shield viral RNAs. Therefore, we provide a model whereby viral IBs escape the antiviral effect of SGs by concealing their own newly synthesized viral RNAs and offer new insights into the emerging role of IBs in viral replication.

Picornavirus 2A protease regulates stress granule formation to facilitate viral translation.

Stress granules (SGs) contain stalled messenger ribonucleoprotein complexes and are related to the regulation of mRNA translation. Picornavirus infection can interfere with the formation of SGs. However, the detailed molecular mechanisms and functions of picornavirus-mediated regulation of SG formation are not clear. Here, we found that the 2A protease of a picornavirus, EV71, induced atypical stress granule (aSG), but not typical stress granule (tSG), formation via cleavage of eIF4GI. Furthermore, 2A was required and sufficient to inhibit tSGs induced by EV71 infection, sodium arsenite, or heat shock. Infection of 2A protease activity-inactivated recombinant EV71 (EV71-2AC110S) failed to induce aSG formation and only induced tSG formation, which is PKR and eIF2α phosphorylation-dependent. By using a Renilla luciferase mRNA reporter system and RNA fluorescence in situ hybridization assay, we found that EV71-induced aSGs were beneficial to viral translation through sequestering only cellular mRNAs, but not viral mRNAs. In addition, we found that the 2A protease of other picornaviruses such as poliovirus and coxsackievirus also induced aSG formation and blocked tSG formation. Taken together, our results demonstrate that, on one hand, EV71 infection induces tSG formation via the PKR-eIF2α pathway, and on the other hand, 2A, but not 3C, blocks tSG formation. Instead, 2A induces aSG formation by cleaving eIF4GI to sequester cellular mRNA but release viral mRNA, thereby facilitating viral translation.

An amino acid of human parainfluenza virus type 3 nucleoprotein is critical for template function and cytoplasmic inclusion body formation.

The nucleoprotein (N) and phosphoprotein (P) interaction of nonsegmented negative-strand RNA viruses is essential for viral replication; this includes N°-P (N°, free of RNA) interaction and the interaction of N-RNA with P. The precise site(s) within N that mediates the N-P interaction and the detailed regulating mechanism, however, are less clear. Using a human parainfluenza virus type 3 (HPIV3) minigenome assay, we found that an N mutant (N(L478A) did not support reporter gene expression. Using in vivo and in vitro coimmunoprecipitation, we found that N(L478A) maintains the ability to form N(L478A)°-P, to self-assemble, and to form N(L478A)-RNA but that N(L478A)-RNA does not interact with P. Using an immunofluorescence assay, we found that N-P interaction provides the minimal requirement for the formation of cytoplasmic inclusion bodies, which contain viral RNA, N, P, and polymerase in HPIV3-infected cells. N(L478A) was unable to form inclusion bodies when coexpressed with P, but the presence of N rescued the ability of N(L478A) to form inclusion bodies and the transcriptional function of N(L478A), thereby suggesting that hetero-oligomers formed by N and N(L478A) are functional and competent to form inclusion bodies. Furthermore, we found that N(L478A) is also defective in virus growth. To our knowledge, we are the first to use a paramyxovirus to identify a precise amino acid within N that is critical for N-RNA and P interaction but not for N(0)-P interaction for the formation of inclusion bodies, which appear to be bona fide sites of RNA synthesis.

Comparative study of Overlap anastomosis and traditional anastomosis in total laparoscopic radical resection of left colon cancer.

PURPOSE: To investigate the safety and feasibility of using linear stapler to complete the side-to-side anastomosis (Overlap method) of distal and proximal colon on taenia coli along the long axis of the intestine in laparoscopic radical resection of left colon cancer. METHODS: From January 2017 to December 2019, the clinical data of 24 patients with total laparoscopic radical resection of left colon cancer and Overlap anastomosis in the general surgery department of Wuhu First People's Hospital were retrospectively analyzed (research group, RG). In addition, 36 patients who underwent laparoscopic-assisted radical resection of left colon cancer during the same period and whose intestinal tubes were removed from the abdominal wall to complete specimen resection and intestinal anastomosis through auxiliary incision were used as controls (control group, CG). The advantages and disadvantages of the two surgical methods were compared through the research indexes during and after the operation. RESULTS: Compared with CG, the total operation time of the patients in RG was shortened (p<0.001), the intraoperative blood loss was less (p<0.001), the abdominal wall incision length was shorter (p<0.001) and the postoperative hospital stay was shorter (p=0.014). There was no significant difference between RG and CG in the number of lymph node dissection, the time of first postoperative anal exhaust and the incidence of postoperative complications (all p>0.05). CONCLUSION: The Overlap anastomosis technique of total laparoscopic radical resection of left colon cancer is feasible and easy to perform. It has the advantages of low incidence of complications, better cosmetic effect and short hospital stay. Although further prospective randomized studies are needed to determine its effects and limitations, it is still recommended that this operation can be popularized in clinical practice.

SG formation relies on eIF4GI-G3BP interaction which is targeted by picornavirus stress antagonists.

Typical stress granules (tSGs) are stalled translation pre-initiation complex aggregations in the cytoplasm, and their formation is a common consequence of translation initiation inhibition under stress. We previously found that 2A protease of picornaviruses blocks tSG formation and induces atypical SG formation, but the molecular mechanism by which 2A inhibits tSG formation remains unclear. Here, we found that eukaryotic translation initiation factor 4 gamma1 (eIF4GI) is critical for tSG formation by interacting with Ras-GTPase-activating protein SH3-domain-binding protein (G3BP), and this interaction is mediated by aa 182-203 of eIF4GI and the RNA-binding domain of G3BP. Upon eIF4GI-G3BP interaction, eIF4GI can assemble into tSGs and rescue tSG formation. Finally, we found that 2A or L protein of picornaviruses blocks tSG formation by disrupting eIF4GI-G3BP interaction. Our findings provide the first evidence that eIF4GI-G3BP interaction is indispensable for tSG formation, and 2A or L protein of picornaviruses interferes eIF4GI-G3BP interaction, thereby blocking tSG formation.

Two second-site mutations compensate the engineered mutation of R7A in vesicular stomatitis virus nucleocapsid protein.

The functional template for the transcription and replication of vesicular stomatitis virus (VSV) is genomic RNA encapsidated by nucleocapsid (N) protein. Previous studies showed that the amino acid R7 in the N-terminal arm of N is involved in N-N interaction in the N-RNA complex. In our study, the recombinant virus with mutation of R7A (rVSV(R7A)) in N was recovered, and the replication level of passage 1 (P1) of rVSV(R7A) was 1000 times lower than that of wild-type rVSV at 37°C. After eight passages, the replication level of P8 of rVSV(R7A) with two second-site mutations in the genome (T242 P in N protein and U7-U8 in G-L gene junction) was significantly higher than that of P1. Furthermore, we demonstrate that the mutation of either T242P or U7-U8 can compensate the effect caused by the mutation of R7A on the replication of rVSV(R7A). Therefore, we conclude that two second-site mutations both can compensated the engineered mutation of R7A in VSV N protein.

Several residues within the N-terminal arm of vesicular stomatitis virus nucleoprotein play a critical role in protecting viral RNA from nuclease digestion.

The nucleoprotein (N) of vesicular stomatitis virus (VSV) plays a central role in transcription and replication by encapsidating genome RNA to form a nucleocapsid as the template for the RNA synthesis. Using minigenome system we evaluated the roles of 21 amino acids of the N-terminal arm of N in forming functional N-RNA templates and found that three triple-amino-acid substitutions (TVK4-6A3, RII7-9A3, and VIV13-15A3) and one single-amino-acid substitution (R7A) resulted in RNA synthesis loss. But all the mutants maintain the ability to oligomerize N, interact with P, and encapsidate viral RNA for template formation. Further analysis showed that the nucleocapsid formed by these mutants failed to protect RNA from nuclease digestion. Then, we found that only recombinant viruses containing R7A could be recovered. Our results show that the several amino acids within the N-terminal arm of N contribute to the template function beyond its role in RNA encapsidation and viral growth.