Large-scale viral outbreaks present significant threats to human health and the stability of our healthcare systems. Such outbreaks are exacerbated by the rapidity with which diseases can spread, facilitated by modern domestic and international travel. In the wake of the recent pandemic, it is likely that the next major viral challenge will be caused by a pathogen with which we have some, albeit limited, familiarity. It is crucial to investigate the biophysical and structural interactions between viruses and their hosts to develop effective therapeutics and vaccines, thereby protecting our most vulnerable populations. We have identified several viruses, monkeypox virus and human astrovirus, that have demonstrated their potential for large-scale circulation but have been historically understudied. Cases of monkeypox virus have been on the rise worldwide since late 2021, and there are currently no vaccines or antiviral against human astroviruses. In this context, I examine the immune evasion mechanisms of the monkeypox virus and the virus-receptor interactions of classical human astroviruses (HAstVs). Poxviruses are double-stranded DNA viruses that cause sores and blisters on infected individuals and are considered contagious among their respective hosts. These viruses have extremely large xiii genomes, averaging 200 open reading frames (ORFs), with the majority of ORFs important for replication and assembly of the virus concentrated in the middle of the genome. The ORFs located at the terminal regions of the genome encode for a broad array of proteins that serve to evade the host immune system. The Fremont lab previously identified a structural scaffold in unrelated poxviral proteins that bore no structural relation to any eukaryotic or prokaryotic protein. Although these proteins share a conserved β-sandwich fold, they appear to offer a diverse range of functions and ligands, all under the umbrella of incapacitating the host immune defenses. This scaffold is called the poxvirus immune evasion (PIE) domain. Monkeypox virus (MPXV) is the causative agent of the smallpox-related disease mpox in humans and has been the cause of recent 2022-2023 and 2024 outbreaks in both endemic and non-endemic countries, including the USA. The smallpox vaccine, which has not been regularly administered since 1980, provided cross-protection against other poxviruses, including MPXV. As such, the majority of people do not have immunity against MPXV, allowing its spread from human-tohuman transmission. MPXV’s immune evasion strategies are not well-studied, and the virus encodes for several PIE proteins. One of these is an ortholog for vCCI, one of the most extensively studied PIE proteins, well-known for its ability to bind to chemokines, which are an important part of anti-viral defense. Here, we characterize the chemokine binding of several PIE proteins from MPXV. We assessed six PIE proteins from MPXV: vCCI, A41, CrmB, M2, Scp-1, and Scp-3. The chemokine binding of these PIEs against 46 chemokines revealed three PIEs capable of forming kinetically stable, long-lasting complexes with multiple chemokines: vCCI, A41, and CrmB. Scp1 and Scp-3 had no specific interaction with any chemokines, while M2 appeared to have a weak signal with two chemokines. We determined that MPXV-encoded vCCI binds its own distinct set xiv of chemokines with nanomolar affinities and long half-lives, while A41 and CrmB had more overlap among the chemokines they bind with a wide range of kinetics. Interestingly, the chemokines that A41/CrmB had the highest affinities for were the same ones that vCCI had the lowest affinities for. However, the varying degree of chemokine binding observed for A41 and CrmB suggests that this might be a vestigial function, and that their primary immunological function is yet to be identified. Still, these results show that MPXV selectively targets different elements of host chemokine networks during its pathogenesis and paves the way to better understand how PIE proteins are important for MPXV vaccine efforts and can even act as immunomodulatory therapeutics. In addition to these studies, we investigated the host receptor(s) for human astroviruses (HAstVs), which has gone unidentified for almost 50 years. Human astroviruses are major causes of gastroenteritis worldwide, especially in children and the elderly. They were initially identified in 1975 in the fecal matter of ill children. They can be broadly divided into two categories: classical (HAstV1-8) and nonclassical (MLB1-3/VA1-5). Seroprevalence studies show that the majority of adults have been infected with at least one HAstV in childhood, although they are extremely likely to be infected with more than one over the course of a lifetime. Additionally, nonclassical HAstVs, which are highly divergent and novel from classical HAstVs, cause neurological complications such as encephalitis and meningitis in immunocompromised individuals. Outbreaks of HAstV happen frequently at daycares, schools, and nursing homes. HAstVs are just one type of astrovirus, and over 20 species can be infected by astroviruses. Additionally, evolutionary analyses suggest that there have been extensive cross-species events of astrovirus between animal species and humans. xv Very little is known about host factors required for HAstV cellular entry. Members of the Baldridge lab utilized complementary CRISPR-Cas9-based knockout and activation screens to identify neonatal Fc receptor (FcRn) and dipeptidyl-peptidase IV (DPP4) as entry factors for HAstV1 and HAstV8 infection of human intestinal epithelial cell lines. Disruption of FcRn or DPP4 using single-guide RNAs reduced HAstV infection in permissive cells. Reciprocally, overexpression of these factors in HEK293, which were previously non-permissive, was sufficient to promote infection. We investigated direct binding between HAstV virions with both entry factors but found that only FcRn had a significant binding signal. We saw this same trend again using purified spike protein against both FcRn and DPP4, and found no binding between HAstV1/8 spike and DPP4. This suggests that FcRn is a receptor for HAstVs while DPP4’s role remains unclear. Finally, inhibitors for DPP4 and FcRn currently in clinical use prevented HAstV1 and HAstV8 infection in cell lines and human enteroids. Thus, our results reveal mechanisms of HAstV entry as well as druggable targets to limit HAstV infection. We biophysically assessed FcRn against spike proteins from classical and nonclassical HAstVs. All spike proteins from classical HAstVs bound to FcRn in a specific and saturable manner, while the nonclassical spikes had little to no interaction with FcRn, suggesting that they may utilize different host factors. Furthermore, there is a ~3-fold increase is binding affinity between HAstV1 and FcRn at pH 5.5, which is the pH of late-endosomal compartments. Lastly, we structurally characterized the interaction between FcRn and mature HAstV1 using cryoEM reconstructions of HAstV1 alone and in complex with FcRn. We determined a 2.77 Å resolution cryo-EM reconstruction of the capsid protein assembled as a trimer. 60 of these trimers organize with T=3 icosahedral symmetry, and feature positively charged coiled-coil bundles at their N-terminal region that project into the core where they likely interact with viral RNA. We xvi found that there are no structural differences in the capsid shell of HAstV1 with or without FcRn. HAstV1 displays 30 dimeric spikes protruding from the 2-fold axes of the mature virion, with deep clefts between the 3- and 5-fold axes, facilitating the protease-mediated removal of the other 60 spike dimers found on the immature virion. We determined a 5 to 9 Å resolution cryo-EM reconstruction of FcRn in complex with HAstV1, the low resolution likely caused by mobility of the spike due to the linker region connecting it to capsid protein. FcRn engages with HAstV1 spikes in a 2:2 stoichiometry, sitting to the sides of the spike dimer. Through competition and mutational assays, we found that FcRn utilizes the same determinants to interact with both HAstV1 spike and IgG. The interaction of FcRn with both IgG and serum albumin is highly pH-dependent, allowing these proteins to be recycled from early endosomes to the plasma membrane for release. In contrast, FcRn binding to the HAstV1 spike is only mildly pH-dependent, potentially providing a mechanism to protect virions from lysosomal degradation as well as plasma membrane release. In summary, these studies reveal how two different viruses, monkeypox virus and human astrovirus, interact with a human host from two different angles: how one evades the host immune system and how one interacts with its entry receptor, respectively. By employing a biophysical approach to these emergent and re-emergent viruses, we enhance our quantitative understanding of their host interactions, thereby providing a more solid foundation for future therapies and vaccines.