The papain-like protease of coronaviruses cleaves ULK1 to disrupt host autophagy

The ongoing pandemic of COVID-19 alongside the outbreaks of SARS in 2003 and MERS in 2012 underscore the significance to understand betacoronaviruses as a global health challenge. SARS-CoV-2, the etiological agent for COVID-19, has infected more than 29 million individuals worldwide with nearly ~1 million fatalities. Understanding how SARS-CoV-2 initiates viral pathogenesis is of the utmost importance for development of antiviral drugs. Autophagy modulators have emerged as potential therapeutic candidates against SARS-CoV-2 but recent clinical setbacks underline the urgent need for better understanding the mechanism of viral subversion of autophagy. Using murine hepatitis virus-A59 (MHV-A59) as a model betacoronavirus, time-course infections revealed a significant loss in the protein level of ULK1, a canonical autophagy regulating serine-threonine kinase, and the concomitant appearance of a possible cleavage fragment. To investigate whether virus-encoded proteases target this protein, we conducted in vitro and cellular cleavage assays and identified ULK1 as a novel bona fide substrate of SARS-CoV-2 papain-like protease (PLpro). Mutagenesis studies discovered that ULK1 is cleaved at a conserved PLpro recognition sequence (LGGG) after G499, separating its N-terminal kinase domain from the C-terminal substrate recognition region. Consistent with this, over-expression of SARS-CoV-2 PLpro is sufficient to impair starvation-induced canonical autophagy and disrupt formation of ULK1-ATG13 complex. Finally, we demonstrated a dual role for ULK1 in MHV-A59 replication, serving a pro-viral functions during early replication that is inactivated at late stages of infection. In conclusion, our study identified a new mechanism by which PLpro of betacoronaviruses induces viral pathogenesis by targeting cellular autophagic pathway (Word count=250) IMPORTANCEThe recent COVID-19 global pandemic alongside the 2003 SARS and 2012 MERS outbreaks underscore an urgent need to better understand betacoronaviruses as pathogens that pose global challenge to human health. Studying the underlying biology of how betacoronaviruses subvert innate cellular defense pathways such as autophagy will help to guide future efforts to develop anti-viral therapy. (Word count= 55)

Evidence of two genetically different lymphotropic herpesviruses present among red deer, sambar, and milu herds in China

Herpesvirus infections in Cervidae are a serious threat affecting some deer species worldwide. In our attempt to identify malignant catarrhal fever-associated herpesviruses in deer herds, ten gammaherpesviral DNA fragments were identified in five species of deer in herds in China by using a pan-herpesvirus polymerase chain reaction assay targeting viral DNA polymerase. Notably, in sambar (Rusa unicolor), a novel gamma-2 herpesvirus was identified that showed a close relationship with fallow deer lymphotropic herpesvirus (LHV), while the other fragments were phylogenetically grouped together with Elk-LHV. Determination of whether these viruses have any clinical implication in these deer species should be undertaken urgently.

Determination of rhein, baicalin and berberine in traditional Chinese medicinal preparations by capillary electrophoresis with two-marker technique.

A CZE method for the identification and determination of three bioactive components, rhein, baicalin and berberine, was developed, with 10 mM borate at pH 9.20 as background electrolyte and direct UV detection at 254 nm. The two-marker (glycyrrhizin acid and cefalexin) technique was used to improve the repeatability of analysis. When the migration indices were used, the repeatability was greatly enhanced compared with migration times. The apparent dissociation contents of rhein and baicalin were also obtained. This method has been successfully applied to the simultaneous analysis of the three components with the recoveries from 96.7% to 104.6%.

Studies on Preparation Technology of ?-cyclodextrin Inclusion Complex of Oleum Viticis Negundo / 中药新药与临床药理

Objective To optimize the preparation technology of ?-cyclodextrin inclusion complex of Oleum Viticis Negundo.Methods Orthogonal test was applied.With the inclusion rate and the content of 1,8-Cineole as the parame ters,L9(34) table was used to examine the effects of 4 factors:the proportion of Oleum Viticis Negundo and ?-cyclodextrin,inclusion temperature,inclusion time and the proportion of Oleum Viticis Negundo and water.Results The best condition was∶oil ∶ ?-cyclodextrin∶ water=1∶8∶70(ml∶g∶ml),stirring for 1.5 hour at 50 ℃.Conclusion Under the optimized condition,inclusion rate of Oleum Viticis Negundo is over 80%.