All results shown are representative of at least three separate experiments. Unpaired student's t-test (two-tailed) or the Mann-Whitney test was used for comparison of two groups where appropriate. Kaplan Meier curve with log-rank test (GraphPad Prism 4.03 software) was used to analyze the mortality rate. pvalue ,0.05 was considered as statistically significant. its protein expression in the liver, spleen and lymph node from PD-1-deficient mice after 72 h of MHV-3 infection in the presence of IFN-c and TNF-a mAbs or rat IgG1 isotype control antibodies was detected by qPCR and immunohistochemistry, respectively. (C) The IFN-c and TNF-a mAbs treatment resulted in decreased damage to the liver, spleen, lymph node and thymus after 72 h of MHV-3 infection. (D) Reduced FGL2 level by IFN-c and TNF-a mAbs treatment resulted in reduced liver damage (indicated by AST and ALT levels). (E) PD-1-deficient mice were infected with MHV-3 (10 PFU) and simultaneously treated with IFN-c and TNF-a blocking mAbs (n = 4) or rat IgG1 control (n = 6), the survival rate was monitored for a total of 15 days. p = 0.025,0.05 was considered significantly different. One representative of three experiments that yielded similar results is shown. Magnification 6 400. NS: not significantly different. *p,0.05 and **p,0.01. n = 5/group. doi:10.1371/journal.ppat.1001347.g007 Figure S3 The number of Foxp3-positive cells was not changed significantly in PD-1-deficient mice after MHV-3 infection. Foxp3-positive cells in the liver, thymus, spleen, and lymph nodes between PD-1-deficient and WT mice at 72 h after MHV-3 infection were detected by immunofluorescence staining (left). Statistical analysis of the number of Foxp3-positive cells in the indicated organs (right). Blue color indicates nuclear DAPI staining. Scale bar = 20mm. NS: not significantly different. Found at: doi:10.1371/journal.ppat.1001347.s003 (1.44 MB TIF) Machupo Virus Glycoprotein Determinants for Human Transferrin Receptor 1 Binding and Cell Entry Machupo virus (MACV) is a highly pathogenic New World arenavirus that causes hemorrhagic fever in humans. MACV, as well as other pathogenic New World arenaviruses, enter cells after their GP1 attachment glycoprotein binds to their cellular receptor, transferrin receptor 1 (TfR1). TfR1 residues essential for this interaction have been described, and a co-crystal of MACV GP1 bound to TfR1 suggests GP1 residues important for this association. We created MACV GP1 variants and tested their effect on TfR1 binding and virus entry to evaluate the functional significance of some of these and additional residues in human and simian cells. We found residues R111, D123, Y122, and F226 to be essential, D155, and P160 important, and D114, S116, D140, and K169 expendable for the GP1-TfR1 interaction and MACV entry. Several MACV GP1 residues that are critical for the interaction with TfR1 are conserved among other New World arenaviruses, indicating a common basis of receptor interaction. Our findings also open avenues for the rational development of viral entry inhibitors. Arenaviruses have single-stranded, bisegmented ambisense RNA genomes and form enveloped virions [1] . Seven arenaviruses cause viral hemorrhagic fever in humans: the Old World arenaviruses Lassa and 'Lujo,' and the New World Clade B arenaviruses Machupo (MACV), Junín (JUNV), Guanarito (GTOV), Sabiá (SABV), and Chapare (CHPV) [2, 3, 4] . All of these viruses are US Select Agents and Risk Group 4 Pathogens [5, 6] . MACV, JUNV, and GTOV are also classified as US National Institute of Allergy and Infectious Disease Category A Priority Pathogens [7] . MACV causes human disease outbreak with high case-fatality rates. To date, at least 1,200 cases with <200 fatalities have been recorded [8, 9] .