In this task, you're given a paragraph from the research paper and your task is to generate a suitable title for the research paper based on the given paper. Under 100 words is a good title length.
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Question: Coronavirus reverse genetic systems have become valuable tools for studying the molecular biology of coronavirus infections. They have been applied to the generation of recombinant coronaviruses, selectable replicon RNAs, and coronavirus-based vectors for heterologous gene expression. Here we provide a collection of protocols for the generation, cloning, and modification of full-length coronavirus cDNA using vaccinia virus as a cloning vector. Based on cloned coronaviral cDNA, we describe the generation of recombinant coronaviruses and stable cell lines containing coronaviral replicon RNAs. Initially, the vaccinia virus-based reverse genetic system was established for the generation of recombinant human coronavirus 229E. However, it is also applicable to the generation of other coronaviruses, such as the avian infectious bronchitis virus, mouse hepatitis virus, and SARS coronavirus.

Answer: Generation of Recombinant Coronaviruses Using Vaccinia Virus as the Cloning Vector and Stable Cell Lines Containing Coronaviral Replicon RNAs


Question: Several polypeptide products of MHV-A59 ORF 1 a were characterized in MHV-A59 infected DBT cells, using antisera directed against fusion proteins encoded in the first 6.5 kb of ORFla. These included the previously identified N-terminal ORF 1 a product, ~28, as well as 290-, 240-, and 50-kDa polypeptides. P28 was always detected as a discrete band without larger precursors, suggesting rapid cleavage of p28 immediately after its synthesis. Once p28 was cleaved there was little degradation of the protein over a 2-hr period. The intracellular cleavage of p28 was not inhibited by the protease inhibitor leupeptin, in contrast to results obtained during in vitro translation of genome RNA (Denison and Perlman, 1986). These data suggest that different protease activities may be responsible for the cleavage of p28 in vitro and in Go. The 290-kDa protein was an intermediate cleavage product derived from a precursor of greater than 400 kDa. The 290-kDa product was subsequently cleaved into secondary products of 50 and 240 kDa. The intracellular cleavage of the 290-kDa polypeptide was inhibited by leupeptin at concentrations which did not inhibit the early cleavage of p28 or the cleavage of the 290-kDa product from its larger polyprotein precursor. In the presence of zinc chloride, a product of >320 kDa was detected, which appears to incorporate p28 at its amino terminus. This suggests that at least two protease activities may be necessary for processing of ORFl a proteins, one of which cleaves p28 and is sensitive to zinc chloride but resistant to leupeptin, and the other which cleaves the 290-kDa precursor and is sensitive to both inhibitors. Both the 290-and 240-kDa proteins should contain sequences predicted to encode two papain-like prOtesS? activities. 0 1992 Academic PVE.S. Inc.

Answer: Intracellular Processing of the N-Terminal ORF la Proteins of the Coronavirus MHV-A59 Requires Multiple Proteolytic Events


Question: Efforts towards developing a vaccine for Middle East respiratory syndrome coronavirus (MERS-CoV) have yielded promising results. Utilizing a variety of platforms, several vaccine approaches have shown efficacy in animal models and begun to enter clinical trials. In this review, we summarize the current progress towards a MERS-CoV vaccine and highlight potential roadblocks identified from previous attempts to generate coronavirus vaccines. Viruses 2019, 11, 74 2 of 17 Preclinical vaccine development for both SARS-CoV and MERS-CoV has largely aimed to stimulate a robust immune response against the viral envelope-protruding spike (S) glycoprotein [28, 29] , a class I fusion protein, and/or the nucleocapsid (N) protein [30, 31] . MERS-CoV S is proteolytically cleaved by host furin [32] during maturation into an S1 domain responsible for binding to DPP4 as well as an S2 domain containing two heptad-repeat regions that facilitate membrane fusion (Figure 1 ). The S1 domain can be further divided into the N-terminal domain (NTD), or S1 A , associated with binding sialic acid [33] , and the receptor binding domain (RBD), comprising the majority of the C-terminal domain of S1. Cryo-electron microscopy studies have shown that the RBD is flexible and opens upward or away from the viral envelope in order to establish contact with DPP4, which may expose S2' [34], a second protease cleavage site within S2. Cleavage at S2' is necessary for membrane fusion upon viral entry [32] . The centrality of S to viral entry helps explain why antibodies that target it are potently neutralizing [35] . On the other hand, while CoV N proteins are abundantly expressed during infection [36] , immunization with SARS-CoV N did not induce strongly neutralizing antibodies [37], likely because N is not displayed on the viral surface. However, N is more conserved than S within CoV lineage [38] , and vaccination with SARS-CoV N was shown to induce cytotoxic T cell responses in mice [39] . Therefore, N may help induce cell-mediated immunity to CoV infection [40] , as may S [41]. CD4+ and CD8+ T cell responses from recovered MERS-CoV patients were particularly strong towards N peptides [42] . However, vaccination with N-based immunogens may carry risks associated with T h 2-related eosinophilic immune enhancement [43] , as may S-based vaccines [44] . Notwithstanding, because of the protection afforded by the robust immune response it generates, S has been the target of most vaccine candidates for MERS-CoV. In this review, we summarize the current state of MERS-CoV vaccine candidates and also describe potential barriers to MERS-CoV vaccine efficacy that first surfaced during research on developing a SARS-CoV vaccine.

Answer:
viruses Middle East Respiratory Syndrome Vaccine Candidates: Cautious Optimism