Argentine hemorrhagic fever is a potentially lethal disease that is caused by Junin virus (JUNV). There are currently around 5 million individuals at risk of infection within endemic regions in Argentina. The live attenuated vaccine strain, Candid #1 (Can), is approved for use in endemic regions and has substantially decreased the number of annual Argentine hemorrhagic fever (AHF) cases. The glycoprotein (GPC) gene is primarily responsible for the attenuation of the Can strain, and we have shown that the absence of an N-linked glycosylation motif in the subunit G1 of the GP complex of Can, which is otherwise present in the wild type pathogenic JUNV, causes GPC retention in the ER. Here, we show that Can GPC aggregates in the ER of infected cells, forming incorrect cross-chain disulfide bonds, which results in impaired GPC processing into G1 and G2. The GPC fails to cleave into its G1 and G2 subunits and is targeted for degradation within lysosomes. Cells infected with the wild-type Romero (Rom) strain do not produce aggregates that are observed in Can infection, and the stress on the ER remains minimal. While the mutation of the N-linked glycosylation motif (T168A) is primarily responsible for the formation of aggregates, other mutations within G1 that occurred earlier in the passage history of the Can strain also contribute to aggregation of the GPC within the ER.ImportanceThe development of vaccines and therapeutics to combat viral hemorrhagic fevers remains a top priority within the Implementation Plan of the U.S. Department of Health and Human Services Public Health Emergency Medical Countermeasures Enterprise. The Can strain, derived from the pathogenic XJ strain of JUNV, has been demonstrated to be both safe and protective against AHF. While the vaccine strain is approved for use in endemic regions within Argentina, the mechanisms of Can attenuation have not been elucidated. A better understanding of the viral genetic determinants of attenuation will improve our understanding of the mechanisms contributing to disease pathogenesis and provide critical information for the rational design of live attenuated vaccine candidates for other viral hemorrhagic fevers.