Junín Virus Infects Mouse Cells and Induces Innate Immune Responses

New world hemorrhagic fever arenaviruses infection of humans results in 15–30% mortality. We performed a high throughput siRNA screen with Junín virus glycoprotein-pseudotyped viruses to find potential host therapeutic targets. Voltage-gated calcium channels (VGCC) subunits, for which there are FDA-approved drugs, were identified in the screen. Knockdown of VGCC subunits or treatment with channel blockers diminished Junín virus-cell fusion and entry into cells and thereby decreased infection. Gabapentin, an FDA-approved drug used to treat neuropathic pain that targets the α2δ2 subunit, inhibited infection of mice by the Candid 1 vaccine strain of the virus. These findings demonstrate that VGCCs play a role in virus infection and have the potential to lead to therapeutic intervention of new world arenavirus infection.

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“…In contrast, only MMTV and not Junín infection was affected by TFR1 knockdown (Fig. S12A), as we previously reported (32). …”

Section: Resultssupporting

confidence: 86%

“…Junín virus can infect mouse cells via a TfR1-independent mechanism, albeit inefficiently compared to human cells (32, 34). Knockdown of CACNA1S or CACNA2D2 greatly decreased infection of mouse macrophage cells by both Junín and MMTV pseudotypes (Fig.…”

Section: Resultsmentioning

confidence: 99%

siRNA Screen for Genes That Affect Junín Virus Entry Uncovers Voltage-Gated Calcium Channels as a Therapeutic Target

et al. 2013

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“…VeroE6 and HEK293T cells were grown in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum. The Candid-1 vaccine strain of JUNV was kindly provided by Robert B. Tesh (University of Texas Medical Branch, Galveston, TX) via Susan R. Ross (University of Pennsylvania, Philadelphia, PA) and was propagated in VeroE6 cells as described previously (14). The antisera used included a monoclonal anti-Flag antiserum (Sigma-Aldrich, St. Louis, MO), a monoclonal anti-JUNV-GP antiserum QC03-BF11 (BEI Resources, Manassas, VA), a monoclonal anti-Tsg101 antiserum (Clone C2; Santa Cruz Biotechnology, Santa Cruz, CA), a rabbit polyclonal anti-EBOV VP40 antiserum described previously (15), and a monoclonal antiactin antiserum (Sigma-Aldrich).…”

Section: Methodsmentioning

confidence: 99%

A Host-Oriented Inhibitor of Junin Argentine Hemorrhagic Fever Virus Egress

Han

Liu

et al. 2014

J Virol

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IMPORTANCE There are currently no FDA-approved vaccines or therapeutics to prevent or treat Argentine hemorrhagic fever (AHF). The causative agent of AHF is Junin virus (JUNV); a New World arenavirus classified as an NIAID/CDC category A priority pathogen.Here, we describe a prototype therapeutic that blocks budding of JUNV and has the potential to function as a broad-spectrum antiviral drug. J unin virus (JUNV) is a New World arenavirus endemic in Argentina that causes severe Argentine hemorrhagic fever and significant mortality in humans (1). The virus is present in excreta from infected rodents and is typically spread to humans via aerosolization (1). JUNV is important because it is a biosafety level 4 pathogen with potential use as a bioterror agent, for which there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics. JUNV is an enveloped, bisegmented negative-sense RNA virus that encodes four proteins: surface glycoprotein precursor (GPC), RNA polymerase (L), nucleoprotein (NP), and small RING finger matrix protein (Z). The Z protein of arenaviruses plays a key role in promoting virus egress and spread and is capable of budding independently from mammalian cells in the form of virus-like particles (VLPs) (2-6). Efficient separation of arenavirus Z VLPs from the plasma membrane is promoted by viral L domains within the Z protein (for a review, see reference 7). L domains function by recruiting host proteins that facilitate efficient virus-cell separation ("pinching-off") (7). JUNV Z protein contains a single PTAP type L domain which interacts with host Tsg101, a component of the endosomal sorting complexes required for transport (ESCRT) machinery (5-7). L-domain-mediated recruitment of host proteins by virus represents a potential target for the development of novel antiviral inhibitors to disrupt virus egress from infected cells to reduce virus dissemination and disease progression (3,6,(8)(9)(10)(11)(12)(13).Based upon the known nuclear magnetic resonance structure of the Tsg101-PTAP interaction site, we conducted an in silico screen for competitive binding inhibitors. We identified several compounds, and the most potent (compound 0013) blocks both Z VLP and live JUNV egress at nanomolar concentrations. Moreover, compound 0013 specifically blocks PTAP-Tsg101 interactions. Intriguingly, since L-domain-containing matrix proteins are generally required for efficient virus-cell separation of emerging RNA viruses (filoviruses, arenaviruses, henipaviruses, rhabdoviruses, paramyxoviruses, and retroviruses), we speculate that inhibitors of this virus-host interaction could represent broadspectrum antiviral drugs against a range of enveloped RNA viruses. MATERIALS AND METHODSCell lines, virus, and antibodies. VeroE6 and HEK293T cells were grown in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum. The Candid-1 vaccine strain of JUNV was kindly provided by Robert B. Tesh (University of Texas Medical Branch, Galveston, TX) via

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“…The first TLR2 agonists identified were bacterial lipoproteins (3). In practice, TLR2 has been reported to recognize a wider range of pathogens than any other TLR, including fungi (4), protozoans (5), worms (6), Mycoplasma (7), Gram-positive and -negative bacteria (8,9), DNA viruses (10), and RNA viruses (11), as well as host molecules such as HMGB1 (12). However, concern is growing that many reported TLR2 agonists are artifacts of possible contamination, cellular debris, or merely molecules that sensitize cells to be activated by authentic TLR2 agonists (13,14).…”

Section: Importancementioning

confidence: 99%

MyD88-Dependent Immunity to a Natural Model of Vaccinia Virus Infection Does Not Involve Toll-Like Receptor 2

Davies

Sei

Siciliano

et al. 2014

J Virol

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Although the pattern recognition receptor Toll-like receptor 2 (TLR2) is typically thought to recognize bacterial components, it has been described to alter the induction of both innate and adaptive immunity to a number of viruses, including vaccinia virus (VACV). However, many pathogens that reportedly encode TLR2 agonists may actually be artifactually contaminated during preparation, possibly with cellular debris or merely with molecules that sensitize cells to be activated by authentic TLR2 agonists. In both humans and mice, the most relevant natural route of infection with VACV is through intradermal infection of the skin. Therefore, we examined the requirement for TLR2 and its signaling adaptor MyD88 in protective immunity to VACV after intradermal infection. We find that although TLR2 may recognize virus preparations in vitro and have a minor role in preventing dissemination of VACV following systemic infection with large doses of virus, it is wholly disposable in both control of virus replication and induction of adaptive immunity following intradermal infection. In contrast, MyD88 is required for efficient induction of CD4 T cell and B cell responses and for local control of virus replication following intradermal infection. However, even MyD88 is not required to induce local inflammation, inflammatory cytokine production, or recruitment of cells that restrict virus from spreading systemically after peripheral infection. Thus, an effective antiviral response does require MyD88, but TLR2 is not required for control of a peripheral VACV infection. These findings emphasize the importance of studying relevant routes of infection when examining innate sensing mechanisms. IMPORTANCEVaccinia virus (VACV) provides the backbone for some of the most widely used and successful viral vaccine vectors and is also related to the human pathogens Cantagalo virus and molluscum contagiosum virus that infect the skin of patients. Therefore, it is vital to understand the mechanisms that induce a strong innate immune response to the virus following dermal infection. Here, we compare the ability of the innate sensing molecule Toll-like receptor 2 (TLR2) and the signaling molecule MyD88 to influence the innate and adaptive immune response to VACV following systemic or dermal infection.

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Junín Virus Infects Mouse Cells and Induces Innate Immune Responses

Cited by 49 publications

References 58 publications

siRNA Screen for Genes That Affect Junín Virus Entry Uncovers Voltage-Gated Calcium Channels as a Therapeutic Target

siRNA Screen for Genes That Affect Junín Virus Entry Uncovers Voltage-Gated Calcium Channels as a Therapeutic Target

A Host-Oriented Inhibitor of Junin Argentine Hemorrhagic Fever Virus Egress

MyD88-Dependent Immunity to a Natural Model of Vaccinia Virus Infection Does Not Involve Toll-Like Receptor 2

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