Decoding the Molecular and Antigenic Interactions in Orthopox Virus Entry

The initial interaction between a virus and a human cell represents the critical first step in viral infection, making its prevention a primary focus in antiviral therapy. The viral entry process remains largely unexploredfor orthopox viruses, which caused an escalating number of outbreaks in recent years. In 2022, the mpox virus (MPXV) clade 2b instigated a multi-country outbreak involving both endemic and non-endemic countries, leading to an international health crisis (WHO) and a public health emergency declaration (U.S.). In 2023, the Democratic Republic of Congo (DRC) experienced an outbreak of the more lethal MPXV clade 1 ( with a fatality rate of up to 10%) linked to sexual transmission. Individual proteins involved in the multi-protein entry/fusion process and targets for neutralizing antibodies were described, yet a comprehensive understanding of orthopox virus entry is missing. This leaves a blind spot in understanding the sequential procedure of viral entry, the intricate interactions among the numerous proteins involved, and the implications for vaccine design and therapies. Here we propose to elucidate the molecular process of orthopox virus entry through side-by-side comparison of MPXV and vaccinia virus (VACV) using an integrative approach that combines methods from virology, proteomics, and immunology. Aim 1: Identifyand characterize the interactions of virus andcellproteins during attachment and entry, and determine antibody accessibility to viral entry proteins. We will conduct in vitro orthopox virus infections of Vero E6 cells to understand interaction processes during viral entry. We will analyze multiple time points within the initial 30 minutes post-infection to track sequential interactions between viral and cellular proteins during the attachment and entry phases. Coupling trifunctional crosslinking of cellular and viral proteins with mass spectrometry will reveal virus–host interactions. We will use RNA interference to confirm the relevance of the identified cellular receptors for orthopox virus entry and infection. We will classify cellular proteins by enrichment analyses and pathway networks. Furthermore, we will use: 1) crosslinking to polyclonal antibodies (purified fromsera of mpox convalescent patients and vaccinees), and 2) quantitative analysis of the biotinylated viral surface proteins to identify antibody-accessible entry proteins on MPXV and VACV virions. Aim 2: Map antigenic properties of the viral proteins from the orthopox virus entry cascade. We will use serum antibodies from mpox convalescent and/or vaccinated individuals, collected during the 2022 NYC mpox outbreak, to study recently reported and newly identified viral proteins of the entry cascade. Together, these approaches will address fundamental questions in orthopox virology and decipher the viral entry process and antibody exposure during entry at the molecular level. The insights gained will provide unprecedented detail to help understand how orthopox viruses infiltrate cells, providing critical knowledge to design next-generation vaccines and treatments.