Date Published: February 6, 2014
Publisher: Public Library of Science
Author(s): Josiah Petersen, Mary Jane Drake, Emily A. Bruce, Amber M. Riblett, Chukwuka A. Didigu, Craig B. Wilen, Nirav Malani, Frances Male, Fang-Hua Lee, Frederic D. Bushman, Sara Cherry, Robert W. Doms, Paul Bates, Kenneth Briley, Christopher F. Basler.
The Bunyaviridae comprise a large family of RNA viruses with worldwide distribution and includes the pathogenic New World hantavirus, Andes virus (ANDV). Host factors needed for hantavirus entry remain largely enigmatic and therapeutics are unavailable. To identify cellular requirements for ANDV infection, we performed two parallel genetic screens. Analysis of a large library of insertionally mutagenized human haploid cells and a siRNA genomic screen converged on components (SREBP-2, SCAP, S1P and S2P) of the sterol regulatory pathway as critically important for infection by ANDV. The significance of this pathway was confirmed using functionally deficient cells, TALEN-mediated gene disruption, RNA interference and pharmacologic inhibition. Disruption of sterol regulatory complex function impaired ANDV internalization without affecting virus binding. Pharmacologic manipulation of cholesterol levels demonstrated that ANDV entry is sensitive to changes in cellular cholesterol and raises the possibility that clinically approved regulators of sterol synthesis may prove useful for combating ANDV infection.
Hantaviruses are a genera of the Bunyaviridae family that includes a large number of human pathogens. Hantaviruses found in the Americas, the so called New World hantaviruses, including Andes virus (ANDV) from Argentina and Chile, can cause a lethal hemorrhagic fever known as hantavirus pulmonary syndrome (HPS) while the Old World hantaviruses from Europe and Asia are associated with Hemorrhagic Fever with Renal Syndrome (HFRS) –. Unlike other members of the Bunyaviridae family, ANDV and the other hantaviruses are not transmitted by arthropod vectors but instead infect humans directly by aerosolized excreta from infected rodents. Entry into host cells by the membrane enveloped hantaviruses depends upon the viral glycoproteins GN and GC, which form a heterodimeric complex on the virion surface following cleavage of a polyprotein precursor –. Although it is clear that hantaviral infection relies upon transit to an acidic intracellular compartment where the viral glycoproteins mediate membrane fusion , , the overall entry process is not fully elucidated.
Viral entry is a complex process often requiring orchestration of protein-protein interactions, cellular signaling, and cellular uptake mechanisms . To begin dissecting this process for ANDV, two independent genetic screens were performed. In the first, insertional mutagenesis was carried out with a gene-trap vector in human haploid cells, a method used previously to identify host cell molecules and pathways used by an array of viral and bacterial pathogens , –. This approach allows near saturation of the human genome, although genes required for cell viability in vitro cannot be interrogated. In the second method, a large-scale RNAi screen provided a complementary approach by producing varying degrees of gene suppression and allowing one to potentially query genes required for cell viability. Previously, the low level extent of overlap in genes discovered in various RNAi screens for the same pathogen has hampered identification of specific requirements –. By employing both of these genetic approaches, we sought to identify cellular pathways important for the early stages of ANDV infection comprehensively. The discovery of components of a cholesterol regulatory complex as an ANDV entry requirement by these two independent screens reinforces the significance of this finding. Moreover, the identification of more than 60 independent insertional mutations in each of the 4 genes of this complex attests to the strength of this observation.