The etiologic agent of COVID-19 is highly contagious and has caused a severe global pandemic. Until now, there has been no simple and reliable system available in a lower-biosafety-grade laboratory for SARS-CoV-2 virologic research and inhibitor screening. In this study, we reported a replicon system which consists of four plasmids expressing the required segments of SARS-CoV-2. Our study revealed that the features for viral RNA synthesis and responses to antivirus drugs of the replicon are similar to those of wild-type viruses. Further analysis indicated that ORF6 provided potent in trans stimulation of the viral replication. Some viral variations, such as 5'UTR-C241T and ORF8-(T28144C) L84S mutation, also exhibit their different impact upon viral replication. Besides, the screening of clinically used drugs identified that several tyrosine kinase inhibitors and DNA-Top II inhibitors potently inhibit the replicon, as well as authentic SARS-CoV-2 viruses. Collectively, this replicon system provides a biosafety-worry-free platform for studying SARS-CoV-2 virology, monitoring the functional impact of viral mutations, and developing viral inhibitors.IMPORTANCE COVID-19 has caused a severe global pandemic. Until now, there has been no simple and reliable system available in a lower-biosafety-grade laboratory for SARS-CoV-2 virologic research and inhibitor screening. We reported a replicon system which consists of four ordinary plasmids expressing the required segments of SARS-CoV-2. Using the replicon system, we developed three application scenarios: (i) to identify the effects of viral proteins on virus replication, (ii) to identify the effects of mutations on viral replication during viral epidemics, and (iii) to perform high-throughput screening of antiviral drugs. Collectively, this replicon system would be useful for virologists to study SARS-CoV-2 virology, for epidemiologists to monitor virus mutations, and for industry to develop antiviral drugs.

The wall teichoic acid (WTA) is a major cell wall component of Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), a common cause of fatal clinical infections in humans. Thus, the indispensable ABC transporter TarGH, which flips WTA from cytoplasm to extracellular space, becomes a promising target of anti-MRSA drugs. Here, we report the 3.9-Å cryo-electron microscopy (cryo-EM) structure of a 50% sequence-identical homolog of TarGH from Alicyclobacillus herbarius at an ATP-free and inward-facing conformation. Structural analysis combined with activity assays enables us to clearly decode the binding site and inhibitory mechanism of the anti-MRSA inhibitor Targocil, which targets TarGH. Moreover, we propose a "crankshaft conrod" mechanism utilized by TarGH, which can be applied to similar ABC transporters that translocate a rather big substrate through relatively subtle conformational changes. These findings provide a structural basis for the rational design and optimization of antibiotics against MRSA.IMPORTANCE The wall teichoic acid (WTA) is a major component of cell wall and a pathogenic factor in methicillin-resistant Staphylococcus aureus (MRSA). The ABC transporter TarGH is indispensable for flipping WTA precursor from cytoplasm to the extracellular space, thus making it a promising drug target for anti-MRSA agents. The 3.9-Å cryo-EM structure of a TarGH homolog helps us to decode the binding site and inhibitory mechanism of a recently reported inhibitor, Targocil, and provides a structural platform for rational design and optimization of potential antibiotics. Moreover, we propose a "crankshaft conrod" mechanism to explain how a big substrate is translocated through subtle conformational changes of type II exporters. These findings advance our understanding of anti-MRSA drug design and ABC transporters.

Human immunodeficiency virus type 1 (HIV-1) can integrate viral DNA into host cell chromosomes to establish a long-term stable latent reservoir, which is a major obstacle to cure HIV-1 infection. The characteristics of the HIV-1 latent reservoir have not been fully understood. Here, we identified 126 upregulated plasma membrane proteins in HIV-1 latently infected cells by a label-free liquid chromatography-tandem mass spectrometry analysis. The higher levels of CD98 expression in multiple HIV-1 latently infected cell lines and primary CD4+ T cells compared to uninfected cells were further confirmed by quantitative reverse transcription PCR (RT-qPCR) and flow cytometry analyses. In addition, CD98high CD4+ T cells displayed hyper-permissiveness to HIV-1 infection and possessed distinct immune phenotypic profiles associated with Th17 and peripheral follicular T helper (pTFH) characteristics. Notably, the CD98high resting memory CD4+ T cells harbored significantly higher cell-associated viral RNA and intact provirus than CD98low counterparts in HIV-1-infected individuals receiving combined antiretroviral therapy. Furthermore, CD98high CD4+ T cells exhibited a robust proliferative capacity and significantly contributed to the clonal expansion of the HIV-1 latent reservoir. Our study demonstrates that CD98 can be used as a novel biomarker of HIV-1 latently infected cells to indicate the effect of various strategies to reduce the viral reservoir. IMPORTANCE Identification of cellular biomarkers is the crucial challenge to eradicate the HIV-1 latent reservoir. In our study, we identified CD98 as a novel plasma membrane biomarker for HIV-1 permissiveness and latent infection. Importantly, CD98high CD4+ T cells exhibited a hyper-permissiveness to HIV-1 infection and significantly contributed to the clonal expansion of the HIV-1 latent reservoir. CD98 could be targeted to develop therapeutic strategies to reduce the HIV-1 latent reservoir in further research.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a global pandemic, which severely endangers public health. Our and others' works have shown that the angiotensin-converting enzyme 2 (ACE2)-containing exosomes (ACE2-exos) have superior antiviral efficacies, especially in response to emerging variants. However, the mechanisms of how the virus counteracts the host and regulates ACE2-exos remain unclear. Here, we identified that SARS-CoV-2 nonstructural protein 6 (NSP6) inhibits the production of ACE2-exos by affecting the protein level of ACE2 as well as tetraspanin-CD63 which is a key factor for exosome biogenesis. We further found that the protein stability of CD63 and ACE2 is maintained by the deubiquitination of proteasome 26S subunit, non-ATPase 12 (PSMD12). NSP6 interacts with PSMD12 and counteracts its function, consequently promoting the degradation of CD63 and ACE2. As a result, NSP6 diminishes the antiviral efficacy of ACE2-exos and facilitates the virus to infect healthy bystander cells. Overall, our study provides a valuable target for the discovery of promising drugs for the treatment of coronavirus disease 2019.