17The coronavirus disease COVID-19, caused by emerging SARS-CoV-2, has posed serious 18 threats to global public health, economic and social stabilities, calling for the prompt 19 development of therapeutics and prophylactics. In this study, we firstly verified that 20 SARS-CoV-2 uses human ACE2 as a cell receptor and its spike (S) protein mediates high 21 membrane fusion activity. Comparing to that of SARS-CoV, the heptad repeat 1 (HR1) 22 sequence in the S2 fusion protein of SARS-CoV-2 possesses markedly increased α-helicity 23 and thermostability, as well as a higher binding affinity with its corresponding heptad repeat 2 24 (HR2) site. Then, we designed a HR2 sequence-based lipopeptide fusion inhibitor, termed 25 IPB02, which showed highly potent activities in inhibiting the SARS-CoV-2 S 26 protein-mediated cell-cell fusion and pseudovirus transduction. IPB02 also inhibited the 27 SARS-CoV pseudovirus efficiently. Moreover, the structure and activity relationship (SAR) 28 of IPB02 was characterized with a panel of truncated lipopeptides, revealing the amino acid 29 motifs critical for its binding and antiviral capacities. Therefore, the presented results have 30 provided important information for understanding the entry pathway of SARS-CoV-2 and the 31 design of antivirals that target the membrane fusion step. 32 33 Keywords: SARS-CoV-2; membrane fusion; fusion inhibitor; lipopeptide 34 35 on June 9, 2020 by guest http://jvi.asm.org/ Downloaded from 3 IMPORTANCE 36The COVID-19 pandemic caused by SARS-CoV-2 presents a serious global public health 37 emergency in urgent need of prophylactic and therapeutic interventions. The S protein of 38 coronaviruses mediates viral receptor-binding and membrane fusion thus being considered a 39 critical target for antivirals. Herein, we report that the SARS-CoV-2 S protein evolves a high 40 activity to mediate cell-cell fusion, significantly differing from the S protein of the previously 41 emerged SARS-CoV. In comparison, the HR1 sequence in the fusion protein of SARS-CoV-2 42 adopts a much higher helical stability and can interact with the HR2 site to form a six-helical 43 bundle structure more efficiently, underlying the mechanism of the enhanced fusion capacity. 44Also importantly, the design of membrane fusion inhibitors with high potencies against both 45 SARS-CoV-2 and SARS-CoV has provided potential arsenals to combat the pandemic and 46 tools to exploit the fusion mechanism. 47 65 homotrimeric class I fusion spike (S) protein to gain entry into host cells (7-9). The S protein 66 comprises of S1 and S2 subunits and exists in a metastable prefusion conformation. The S1 67 subunit, which contains a receptor-binding domain (RBD) capable of functional folding 68 independently, is responsible for virus binding to the cell surface receptor. A recent study 69 on June 9, 2020 by guest http://jvi.asm.org/ Downloaded from 5suggested that ACE2-binding affinity of the RBD of SARS-CoV-2 is up to 20-fold higher 70 than that of SARS-CoV, which may contribute to the significantl...

Background:We recently found that N-terminal residues Met-626 and Thr-627 of HIV-1 fusion inhibitor CP621-652 adopt a unique hook-like structure, termed the M-T hook. Results: The structure and function of the M-T hook have been characterized. Conclusion: The M-T hook is critical for the stability and antiviral activity of HIV-1 fusion inhibitors. Significance: Our data provide important information for designing novel HIV-1 fusion inhibitors.

Peptides derived from the C-terminal heptad repeat (C peptides) of HIV-1 gp41 are potent inhibitors against virus entry. However, development of a short C peptide possessing high anti-HIV potency is considered a daunting challenge. We recently discovered that the residues Met626 and Thr627 preceding the pocket-binding domain of the C peptide adopt a unique M-T hook structure that is crucial for the design of HIV-1 fusion inhibitors. In this study, we first presented a proof-of-concept prototype that the M-T hook residues can dramatically improve the antiviral activity and thermostability of a short C peptide. We then generated a 24-mer peptide termed MT-SC22EK by incorporating the M-T hook structure to the N terminus of the poorly active short C peptide SC22EK. Amazingly, MT-SC22EK inhibited HIV-1-mediated cell fusion and infection at a level comparable to C34, T1249, SC29EK, and sifuvirtide, and it was highly active against diverse HIV-1 subtypes and variants, including those T20 (enfuvirtide) and SC29EK-resistant viruses. The high-resolution crystal structure of MT-SC22EK reveals the N-terminal M-T hook conformation folded by incorporated Met626 and Thr627 and identifies the C-terminal boundary critical for the anti-HIV activity. Collectively, our studies provide new insights into the mechanisms of HIV-1 fusion and its inhibition.

LP-11 has high potentials for clinical development and it can serve as an ideal tool for exploring the mechanisms of HIV-1 fusion and inhibition.

Infection with hepatitis E virus (HEV) may be diagnosed by the presence of HEV RNA or anti-HEV antibodies. An enzyme immunoassay (EIA) was developed for the detection of antigen. Twenty-four monoclonal antibodies (mAbs) were produced. An indirect sandwich EIA was developed to detect HEV antigen using a combination of three mAbs as coating antibodies. Approximately 44.6% (33/74), 28.6% (50/175), and none (0/27) of sera positive for anti-HEV IgM alone, both anti-HEV IgM and IgG, and anti-HEV IgG alone also were positive for HEV antigen using this EIA. Forty-two HEV antibody-positive sera were tested for HEV RNA and antigen in parallel and the concordance was 81.0% (34/42). All PCR products were found to belong to HEV genotype 4. In order to evaluate the temporal relationship between HEV antigen positivity and HEV RNA, anti-HEV IgG and IgM, and ALT concentrations, macaques were infected with HEV genotypes 1 and 4 and serial samples were collected. The results showed that the antigen EIA can detect the capsid proteins of both genotypes. HEV antigen was detectable prior to ALT elevation and the appearance of anti-HEV antibodies in the infected monkeys and lasted for several weeks in all cases. HEV antigen became detectable in the serum at almost the same time as HEV RNA in feces but persisted for 4 weeks less than HEV RNA. This assay should be valuable for the diagnosis of acute hepatitis E, particularly in the window period prior to seroconversion to anti-HEV.