Wnt3a Facilitates SARS-CoV-2 Pseudovirus Entry into Cells.

How ACE2 functions as the major host receptor of SARS-CoV-2 despite having low expression in the lungs is still unknown. To facilitate the development of therapeutic strategies against coronaviruses, gaining a deeper comprehension of the molecular mechanism of SARS-CoV-2 infection is imperative. In our previous study, we identified several potential host factors of SARS-CoV-2 using an shRNA arrayed screen, one of which was Wnt3a. Here, we validated the significance of Wnt3a, a potent activator of the Wnt/ß-catenin signaling pathway, for SARS-CoV-2 entry into cells by evaluating the effects of its knockdown and overexpression on SARS-CoV-2 pseudotyped virus entry. Further analysis revealed that SARS-CoV-2 pseudotyped virus infection activates the canonical Wnt/ß-catenin signaling pathway, which we found could subsequently stimulate ACE2 transcription. Collectively, our study identified Wnt3a as an important host factor that facilitates ACE2-mediated virus infection. Insight into the virus entry mechanism is impactful as it will aid in developing novel therapeutic strategies against current and future coronavirus pandemics.

Sustained chronic inflammation and altered childhood vaccine responses in children exposed to Zika virus.

BACKGROUND: Congenital Zika virus (ZIKV) infection leads to severe newborn abnormalities, but its long-term impact on childhood immunity is not well understood. This study aims to investigate the serum proteomics in children exposed to ZIKV during pregnancy to understand potential immunological consequences during early childhood. METHODS: The study included ZIKV-exposed infants (ZEI) at birth (n = 42) and children exposed to ZIKV (ZEC) at two years of age (n = 20) exposed to ZIKV during pregnancy, as well as healthy controls. Serum proteomic analysis was performed on these groups to assess inflammation and immune profiles. Additionally, antibody titres against two common childhood vaccines, DTaP and MMR, were measured in healthy controls (n = 50) and ZEC (n = 92) to evaluate vaccine-induced immunity. FINDINGS: Results showed elevated inflammation in ZEI with birth abnormalities. Among ZEC, despite most having normal clinical outcomes at two years, their serum proteomics indicated a bias towards Th1-mediated immune responses. Notably, ZEC displayed reduced anti-Diphtheria toxin and anti-Clostridium tetani IgG levels against DTaP and MMR vaccines. They also exhibited lower antibody titres particularly against Th2-biased DTaP vaccines, but not Th1-biased MMR vaccines. INTERPRETATION: In conclusion, the study highlights the long-term immunological consequences of congenital ZIKV exposure. Heightened inflammation was observed in ZEI with abnormalities at birth, while ZEC maintained a chronic Th1-biased immune profile. The impaired response to Th2-biased vaccines raises concerns about lasting effects of ZIKV exposure on immune responses. Consequently, there is a need for continued longitudinal clinical monitoring to identify potential immune-related complications arising from prenatal exposure to ZIKV. FUNDING: This work was partially funded by the National Institute of Allergy and Infectious Diseases (NIAID) and National Institute of Dental and Craniofacial Research (NIDCR).

Characterization of host substrates of SARS-CoV-2 main protease.

The main protease (Mpro) plays a crucial role in coronavirus, as it cleaves viral polyproteins and host cellular proteins to ensure successful replication. In this review, we discuss the preference in the recognition sequence of Mpro based on sequence-based studies and structural information and highlight the recent advances in computational and experimental approaches that have aided in discovering novel Mpro substrates. In addition, we provide an overview of the current understanding of Mpro host substrates and their implications for viral replication and pathogenesis. As Mpro has emerged as a promising target for the development of antiviral drugs, further insight into its substrate specificity may contribute to the design of specific inhibitors.

A disintegrin and metalloproteinase domain 9 facilitates SARS-CoV-2 entry into cells with low ACE2 expression.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the Coronavirus disease-19 (COVID-19) pandemic, utilizes angiotensin-converting enzyme 2 (ACE2) as a receptor for virus infection. However, the expression pattern of ACE2 does not coincide with the tissue tropism of SARS-CoV-2, hinting that other host proteins might be involved in facilitating SARS-CoV-2 entry. To explore potential host factors for SARS-CoV-2 entry, we performed an arrayed shRNA screen in H1650 and HEK293T cells. Here, we identified a disintegrin and a metalloproteinase domain 9 (ADAM9) protein as an important host factor for SARS-CoV-2 entry. Our data showed that silencing ADAM9 reduced virus entry, while its overexpression promoted infection. The knockdown of ADAM9 decreased the infectivity of the variants of concern tested-B.1.1.7 (alpha), B.1.617.2 (delta), and B.1.1.529 (omicron). Furthermore, mechanistic studies indicated that ADAM9 is involved in the binding and endocytosis stages of SARS-CoV-2 entry. Through immunoprecipitation experiments, we demonstrated that ADAM9 binds to the S1 subunit of the SARS-CoV-2 Spike. Additionally, ADAM9 can interact with ACE2, and co-expression of both proteins markedly enhances virus infection. Moreover, the enzymatic activity of ADAM9 facilitates virus entry. Our study reveals an insight into the mechanism of SARS-CoV-2 virus entry and elucidates the role of ADAM9 in virus infection. IMPORTANCE COVID-19, an infectious respiratory disease caused by SARS-CoV-2, has greatly impacted global public health and the economy. Extensive vaccination efforts have been launched worldwide over the last couple of years. However, several variants of concern that reduce the efficacy of vaccines have kept emerging. Thereby, further understanding of the mechanism of SARS-CoV-2 entry is indispensable, which will allow the development of an effective antiviral strategy. Here, we identify a disintegrin and metalloproteinase domain 9 (ADAM9) protein as a co-factor of ACE2 important for SARS-CoV-2 entry, even for the variants of concern, and show that ADAM9 interacts with Spike to aid virus entry. This virus-host interaction could be exploited to develop novel therapeutics against COVID-19.

Functional assessments of SARS-CoV-2 single-round infectious particles with variant-specific spike proteins on infectivity, drug sensitivity, and antibody neutralization.

Working with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is restricted to biosafety level III (BSL-3) laboratory. The study used a trans-complementation system consisting of virus-like particles (VLPs) and DNA-launched replicons to generate SARS-CoV-2 single-round infectious particles (SRIPs) with variant-specific spike (S) proteins. S gene of Wuhan-Hu-1 strain (SWH1) or Omicron BA.1 variant (SBA.1), along with the envelope (E) and membrane (M) genes, were cloned into a tricistronic vector, co-expressed in the cells to produce variant-specific S-VLPs. Additionally, the replicon of the WH1-like strain without S, E, M and accessory genes, was engineered under the control by a CMV promoter to produce self-replicating RNAs within VLP-producing cells, led to create SWH1- and SBA.1-based SARS-CoV-2 SRIPs. The SBA.1-based SRIP showed lower virus yield, replication, N protein expression, fusogenicity, and infectivity compared to SWH1-based SRIPs. SBA.1-based SRIP also exhibited intermediate resistance to neutralizing antibodies produced by SWH1-based vaccines, but were effective at infecting cells with low ACE2 expression. Importantly, both S-based SRIPs responded similarly to remdesivir and GC376, with EC50 values ranging from 0.17 to 1.46 µM, respectively. The study demonstrated that this trans-complementation system is a reliable and efficient tool for generating SARS-CoV-2 SRIPs with variant-specific S proteins. SARS-CoV-2 SRIPs, mimicking authentic live viruses, facilitate comprehensive analysis of variant-specific virological characteristics, including antibody neutralization, and drug sensitivity in non-BSL-3 laboratories.

Effects of Basic Amino Acids and Their Derivatives on SARS-CoV-2 and Influenza-A Virus Infection.

Amino acids have been implicated with virus infection and replication. Here, we demonstrate the effects of two basic amino acids, arginine and lysine, and their ester derivatives on infection of two enveloped viruses, SARS-CoV-2, and influenza A virus. We found that lysine and its ester derivative can efficiently block infection of both viruses in vitro. Furthermore, the arginine ester derivative caused a significant boost in virus infection. Studies on their mechanism of action revealed that the compounds potentially disturb virus uncoating rather than virus attachment and endosomal acidification. Our findings suggest that lysine supplementation and the reduction of arginine-rich food intake can be considered as prophylactic and therapeutic regimens against these viruses while also providing a paradigm for the development of broad-spectrum antivirals.