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Breaking down arenavirus cell entry

Periodic Reporting for period 1 - BRACE (Breaking down arenavirus cell entry)

Reporting period: 2015-10-01 to 2017-09-30

BACKGROUND & IMPORTANCE:
Arenaviruses represent a fast-growing group of hemorrhagic fever viruses that include lethal human pathogens such as Lassa virus, Machupo virus, Junin virus, Guanarito virus, Sabia virus and Lujo virus. Lassa virus is endemic in West Africa causing ~500.000 cases on a yearly basis. The other viruses can cause South American hemorrhagic fever, with cases occurring annually in a constantly expanding endemic region. Lujo virus has been associated with an outbreak of hemorrhagic fever in South Africa. The mortality rate ranges from 15-30%. With the exception of Junin virus, no FDA-approved vaccines are available and with limited alternative therapeutic options, identification of new drug targets for the development of effective antivirals is urgently needed. Infectious diseases continue to be one of the largest contributors to morbidity and mortality in the world today. This is again demonstrated by the 2014 outbreak of Ebola virus in Western Africa, which up until now has caused ~2,000 deaths with many more people being infected. Without effective vaccines and antivirals available, these epidemics will continue to be a significant health problem plus the economic costs of such outbreaks are often out of proportion to their death toll. In addition, the continuing growth of global travel favours the spread of infectious diseases. The efficiency and range of modern transport systems puts people at risk from the emergence of both familiar and new infectious diseases. Therefore it is important to keep searching for new and better targets for antiviral drugs so that we can combat these pathogens.

OBJECTIVES:
The main goal of this project was to identify new cell entry factors for arenaviruses, with a particular interest in virus receptors. To this end, viral reverse genetics was combined with haploid genetics in human cells, a methodology that has been effectively implemented during the last 4 years to study entry of different viruses. We created a comprehensive overview of host factors that are important for arenavirus entry into cells, resulting in a list of human genes that encode candidate arenavirus receptors. Especially for Lujo virus several cell-surface localized receptor candidates could be identified. Follow-up work lead to the validation of those selected genes by creating knockout cells for each individual gene and determine their susceptibility to infection. Genes that, upon inactivation, caused potent resistance to infection were further studied. The proteins encoded by these genes were expressed and investigated for their ability to bind arenavirus envelope proteins. For the proteins that interacted, the internalization pathway in context of infection was analysed in further detail. All together, this lead to mechanistic insight in arenavirus entry pathways in human cells and pinpointed new targets for much-needed antiviral therapies against some of these hemorrhagic fever viruses.

CONCLUSIONS:
Using the technology described above, we have uncovered many host factors for several arenavirus family members. Most importantly, we have identified, amongst others, a novel, specific cell entry receptor for Lujo virus. These insights provide a novel target for therapeutic intervention.
Several different VSVs with different arenavirus glycoproteins were recovered, amplified and tested for their infectivity on HAP1 cells. VSVs with the glycoproteins of GTOV, JUNV, LUJV, MACV were plaque-purified twice and amplified in Vero-E6 cells. Virus stocks were generated that have sufficiently high titres to infect HAP1 cells, thereby enabling their use in genetic screens. For all the viruses that were amplified, appropriate screening conditions were established and large batches of mutagenized cells were generated. Mutagenized cell populations were independently selected with VSV-LUJV, VSV-GTOV, VSV-JUNV, and VSV-MACV and resistant cell populations were obtained. Deep sequencing of the mutations in the resistant cell populations was performed for each individual virus selection. The large datasets from four different virus screens were systematically analysed in order to create an overview of host factors.

Several potential receptor candidate genes were identified for all the viruses that were subjected to haploid genetic screening. Especially for LUJV several potential receptor genes could be identified (3 major hits that contain a transmembrane protein [i.e. TMEM30A, CD63 and NRP2]). Knockouts in several different cell lines were created using CRISPR/Cas9-mediated gene editing and candidate receptor genes were cloned into mammalian expression vectors, including lentiviral expression cassettes. From these genes, NRP2 could be identified as a bona fide entry receptor for LUJV. Together with NRP2, CD63 (facilitating membrane fusion mediated by the LUJV glycoprotein) was also identified as a potential drug target for anti-LUJV therapy.

The entry pathway of LUJV was studied in more detail. Fluorescent versions of NRP2 and CD63 were constructed and placed into cells using lentiviral delivery. After functional conformation, we could show that LUJV viral particles co-localize with CD63 after entry into cells and that the entry pathway is sensitive to drugs that inhibit the vacuolar lysosomal ATPase, indicating that LUJV cell entry is dependent on low pH. Overall, we created a comprehensive overview of LUJV cell entry that is distinct from the entry pathways that are hijacked by other arenaviruses.

The LUJV cell entry story was published in Cell Host & Microbe in November 2017. We anticipate that this story will be presented at several different international conferences in the near future.
We also anticipate that data generated during
The work performed in this project provided insights into the complex cell entry strategies of arenaviruses, especially for Lujo virus. Lujo virus has been so far responsible only for a very limited number of cases of lethal hemorraghic fever, but because its high morbidity and lethality there is a great concern about its possible re-emergence and wider dissemination with a corresponding increase in human cases of Lujo virus-associated disease. Our discovery of NRP2 as an entry receptor for Lujo virus could trigger the development of antivirals/antibodies targeting this receptor requirement and has the potential to limit spread of the disease if the virus would re-emerge.
Cell entry pathway of Lujo virus