Abstract

Arenaviruses are enveloped, negative-sense RNA viruses maintained in rodent reservoirs and transmitted to humans through exposure to aerosolized rodent excreta, where they can cause severe and sometimes fatal disease. The geographic distribution of arenaviruses reflects the range of their rodent reservoirs, resulting in the classification into Old World and New World groups, with New World arenaviruses further subdivided into clades A–D. Viral entry is the first step of infection and a major determinant of host range, tissue tropism, and zoonotic potential. Further, viral entry is a tightly regulated, multistep process that couple’s receptor engagement with intracellular trafficking and pH-dependent membrane fusion within the endolysosomal network. For arenaviruses, entry is mediated by the viral glycoprotein complex (GPC), which consists of the receptor-binding subunit (GP1), the fusion subunit (GP2), and a retained stable signal peptide (SSP) that plays a critical regulatory role in fusion activation. Comparative studies across arenavirus species have revealed that differences in receptor usage and fusion activation correlate with distinct endosomal requirements, underscoring the importance of host factor identity in shaping viral entry mechanisms. Some Old World arenaviruses, such as Lassa virus and lymphocytic choriomeningitis virus, bind α-dystroglycan at the plasma membrane and subsequently undergo an acidic pH-induced receptor switch to endolysosomal host factors, Lysosomal-associated protein 1 (LAMP1) and CD164 respectively, to facilitate viral fusion. Pathogenic clade B New World arenaviruses, including Junín and Machupo viruses, utilize human transferrin receptor 1 as a primary plasma membrane receptor. In these viruses, low pH has been considered sufficient to trigger membrane fusion, and no endolysosomal receptors have been defined. However, other New World arenaviruses, such as clade A, do not efficiently engage Transferrin Receptor 1 or α-dystroglycan, and entry receptors have remained undefined. Suggesting that New World Arenaviruses might employ alternative host factors and distinct entry strategies that more closely resemble the OW arenavirus receptor switching paradigm rather than the hTfR1-dependent pathway of clade B New Worlds. To identify host determinants required for New World arenavirus entry, I generated a panel of replication-competent chimeric vesicular stomatitis viruses encoding glycoproteins from representative New World arenaviruses and performed genome-wide loss-of-function CRISPR screens. These screens identified established endolysosomal factors as well as previously unrecognized host determinants. Among these, CD164 emerged as a critical host factor for infection by multiple clade A New World arenaviruses, including Pichindé, Paraná, and Flexal viruses. Demonstrated by genetic ablation of CD164, which markedly reduced infection, and susceptibility was restored by complementation. Mechanistic studies demonstrated that Pichindé virus (PICV) GP1 binds directly and specifically to the cysteine-rich domain (CRD) of CD164 in a pH-dependent manner. Structural modeling, deep mutational scanning, and targeted mutagenesis demonstrated that receptor recognition is mediated through conserved main-chain interactions between a β-sheet within PICV GP1 and the C-terminus of the CD164 CRD. Further, acid-bypass and fusion assays show that CD164 and acidic pH are both required for GPC mediated fusion. Supporting a model in which CD164 acts as an endolysosomal receptor and engagement of the CRD by GP1 promotes membrane fusion and cytoplasmic delivery of viral contents. To determine whether dependence on CD164 could be bypassed, PICV GPC-bearing chimeric VSV was passaged on CD164-deficient cells. Escape variants arose after a single passage through mutations in GP2 and SSP rather than GP1. A dominant mutation in GP2 (T354N) lowered the pH threshold for fusion activation and partially restored infection in the absence of CD164. Predictive structural modeling indicated that mutations in GP2 induce allosteric changes in GP1 and the overall GPC architecture supporting the hypothesis that these mutations facilitate GPC destabilization. These findings support a model in which GP1 engagement of CD164 causes GPC destabilization, facilitating GP2 activation, and in which increased acid sensitivity through mutations in GP2 can partially compensate for the absence of receptor engagement. Together, this work identifies host factors co-opted during New World arenavirus entry, establishes CD164 as an endolysosomal host factor required for clade A New World arenaviruses entry and fusion, and promotes a model by which receptor engagement, or more acid labile mutations, promote GP2-mediated membrane fusion. These findings expand the conceptual framework of arenavirus entry and further expands the receptor switching paradigm to New World arenaviruses.

Committee Chair

Sean Whelan

Committee Members

Carolina López; Daved Fremont; Megan Baldridge; Michael Diamond

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

4-27-2026

Language

English (en)

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Virology Commons

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