Vibor Laketa
Profile Url: vibor-laketa
Researcher at Center for Infectious Diseases, Heidelberg University Hospital, Heidelberg, Germany
Combinations of direct-acting antivirals are needed to minimize drug-resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) and testing of a number of drug regimens has led to conflicting results. Here we show that cobicistat, which is an-FDA approved drug-booster that blocks the activity of the drug metabolizing proteins Cytochrome P450-3As (CYP3As) and P-glycoprotein (P-gp), inhibits SARS-CoV-2 replication. Cell-to-cell membrane fusion assays indicated that the antiviral effect of cobicistat is exerted through inhibition of spike protein-mediated membrane fusion. In line with this, incubation with low micromolar concentrations of cobicistat decreased viral replication in three different cell lines including cells of lung and gut origin. When cobicistat was used in combination with the putative CYP3A target and nucleoside analog remdesivir, a synergistic effect on the inhibition of viral replication was observed in cell lines and in a primary human colon organoid. The cobicistat/remdesivir combination was able to potently abate viral replication to levels comparable to mock-infected cells leading to an almost complete rescue of infected cell viability. These data highlight cobicistat as a therapeutic candidate for treating SARS-CoV-2 infection and as a potential building block of combination therapies for COVID-19.
Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of that are flaviviruses, such as dengue virus and Zika virus that cause millions of yearly infections and spread around the globe, and coronaviruses, such as SARS-CoV-2, which is the cause of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of virology research in determining mechanisms to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective interventions. Here, we describe the generation and characterization of a reporter system to visualize dengue virus and SARS-CoV-2 replication in live cells. The system is based on viral protease activity causing cleavage and nuclear translocation of an engineered fluorescent protein that is expressed in the infected cells. We show the suitability of the system for live cell imaging and visualization of single infected cells as well as for screening and testing of antiviral compounds. Given the modular building blocks, the system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility. IMPORTANCE Reporter systems are useful tools for fast and quantitative visualization of viral replication and spread within a host cell population. Here we describe a reporter system that takes advantage of virus-encoded proteases that are expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the fluorescent protein translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.
Emergence of the novel pathogenic coronavirus SARS-CoV-2 and its rapid pandemic spread presents numerous questions and challenges that demand immediate attention. Among these is the urgent need for a better understanding of humoral immune response against the virus as a basis for developing public health strategies to control viral spread. For this, sensitive, specific and quantitative serological assays are required. Here we describe the development of a semi-quantitative high-content microscopy-based assay for detection of three major classes (IgG, IgA and IgM) of SARS-CoV-2 specific antibodies in human samples. The possibility to detect antibodies against the entire viral proteome together with a robust semi-automated image analysis workflow resulted in specific, sensitive and unbiased assay which complements the portfolio of SARS-CoV-2 serological assays. The procedure described here has been used for clinical studies and provides a general framework for the application of quantitative high-throughput microscopy to rapidly develop serological assays for emerging virus infections. ### Competing Interest Statement The authors have declared no competing interest.