Search Results (5)

Arenaviruses include important zoonotic pathogens that cause hemorrhagic fever (e.g., Junín virus; JUNV) as well as other viruses that are closely related but apathogenic (e.g., Tacaribe virus; TCRV). We have found that, while TCRV and JUNV differ in their ability to induce apoptosis in infected cells, due to active inhibition of caspase activation by the JUNV nucleoprotein, both viruses trigger similar upstream pro-apoptotic signaling events, including the activation/phosphorylation of p53. In the case of TCRV, the pro-apoptotic factor Bad is also phosphorylated (leading to its inactivation). These events clearly implicate upstream kinases in regulating the induction of apoptosis. Consistent with this, here we show activation in TCRV-infected cells of the stress-activated protein kinases p38 and JNK, which are known to regulate p53 activation, as well as the downstream kinase MK2 and transcription factor c-Jun. We also observed the early transient activation of Akt, but not Erk. Importantly, the chemical inhibition of Akt, p38, JNK and c-Jun all dramatically reduced viral growth, even though we have shown that inhibition of apoptosis itself does not. This indicates that kinase activation is crucial for viral infection, independent of its downstream role in apoptosis regulation, a finding that has the potential to shed further light on the determinants of arenavirus pathogenesis, as well as to inform future therapeutic approaches. Full article

Replication-competent reporter-expressing viruses are crucial tools in molecular virology with applications that range from antiviral screening to live-cell imaging of protein spatiotemporal dynamics. However, there is currently little information available regarding viable strategies to develop reporter-expressing arenaviruses. To address this, we used Tacaribe virus (TCRV), an apathogenic BSL2 arenavirus, to assess the feasibility of different reporter expression approaches. We first generated trisegmented TCRV viruses with either the glycoprotein (GP) or nucleoprotein (NP) replaced by a reporter (GFP, mCherry, or nanoluciferase). These viruses were all viable, but showed marked differences in brightness and attenuation. Next, we generated terminal fusions with each of the TCRV proteins (i.e., NP, GP, polymerase (L), matrix protein (Z)) either with or without a T2A self-cleavage site. We tested both the function of the reporter-fused proteins alone, and the viability of corresponding recombinant TCRVs. We successfully rescued viruses with both direct and cleavable reporter fusions at the C-terminus of Z, as well as cleavable N-terminal fusions with NP. These viruses all displayed detectable reporter activity, but were also moderately attenuated. Finally, reporter proteins were inserted into a flexible hinge region within L. These viruses were also viable and showed moderate attenuation; however, reporter expression was only detectable for the luminescent virus. These strategies provide an exciting range of new tools for research into the molecular biology of TCRV that can likely also be adapted to other arenaviruses. Full article

A specialized and fine-tuned immune response of bats upon infection with viruses is believed to provide the basis for a “friendly” coexistence with these pathogens, which are often lethal for humans and other mammals. First insights into the immunity of bats suggest that bats have evolved to possess their own strategies to cope with viral infections. Yet, the molecular details for this innocuous coexistence remain poorly described and bat infection models are the key to unveiling these secrets. In Jamaican fruit bats (Artibeus jamaicensis), a New World bat species, infection experiments with its (putative) natural viral pathogens Tacaribe virus (TCRV), rabies virus (RABV), and the bat influenza A virus (IAV) H18N11, have contributed to an accurate, though still incomplete, representation of the bat-imposed immunity. Surprisingly, though many aspects of their innate and adaptive immune responses differ from that of the human immune response, such as a contraction of the IFN locus and reduction in the number of immunoglobulin subclasses, variations could also be observed between Jamaican fruit bats and other bat species. Full article

The New World (NW) mammarenavirus group includes several zoonotic highly pathogenic viruses, such as Junin (JUNV) or Machupo (MACV). Contrary to the Old World mammarenavirus group, these viruses are not able to completely suppress the innate immune response and trigger a robust interferon (IFN)-I response via retinoic acid-inducible gene I (RIG-I). Nevertheless, pathogenic NW mammarenaviruses trigger a weaker IFN response than their nonpathogenic relatives do. RIG-I activation leads to upregulation of a plethora of IFN-stimulated genes (ISGs), which exert a characteristic antiviral effect either as lone effectors, or resulting from the combination with other ISGs or cellular factors. The dsRNA sensor protein kinase receptor (PKR) is an ISG that plays a pivotal role in the control of the mammarenavirus infection. In addition to its well-known protein synthesis inhibition, PKR further modulates the overall IFN-I response against different viruses, including mammarenaviruses. For this study, we employed Tacaribe virus (TCRV), the closest relative of the human pathogenic JUNV. Our findings indicate that PKR does not only increase IFN-I expression against TCRV infection, but also affects the kinetic expression and the extent of induction of Mx1 and ISG15 at both levels, mRNA and protein expression. Moreover, TCRV fails to suppress the effect of activated PKR, resulting in the inhibition of a viral titer. Here, we provide original evidence of the specific immunomodulatory role of PKR over selected ISGs, altering the dynamic of the innate immune response course against TCRV. The mechanisms for innate immune evasion are key for the emergence and adaptation of human pathogenic arenaviruses, and highly pathogenic mammarenaviruses, such as JUNV or MACV, trigger a weaker IFN response than nonpathogenic mammarenaviruses. Within the innate immune response context, PKR plays an important role in sensing and restricting the infection of TCRV virus. Although the mechanism of PKR for protein synthesis inhibition is well described, its immunomodulatory role is less understood. Our present findings further characterize the innate immune response in the absence of PKR, unveiling the role of PKR in defining the ISG profile after viral infection. Moreover, TCRV fails to suppress activated PKR, resulting in viral progeny production inhibition. Full article

Mammarenaviruses are enveloped and segmented negative-stranded RNA viruses that comprise several pathogenic members associated with severe human hemorrhagic fevers. Tacaribe virus (TCRV) is the prototype for the New World group of mammarenaviruses and is not only naturally attenuated but also phylogenetically and antigenically related to all South American pathogenic mammarenaviruses, particularly the Junín virus (JUNV), which is the etiological agent of Argentinian hemorrhagic fever (AHF). Moreover, since TCRV protects guinea pigs and non-human primates from lethal challenges with pathogenic strains of JUNV, it has already been considered as a potential live-attenuated virus vaccine candidate against AHF. Here, we report the development of a reverse genetic system that relies on T7 polymerase-driven intracellular expression of the complementary copy (antigenome) of both viral S and L RNA segments. Using this approach, we successfully recovered recombinant TCRV (rTCRV) that displayed growth properties resembling those of authentic TCRV. We also generated a chimeric recombinant TCRV expressing the JUNV glycoproteins, which propagated similarly to wild-type rTCRV. Moreover, a controlled modification within the S RNA 5′ non-coding terminal sequence diminished rTCRV propagation in a cell-type dependent manner, giving rise to new perspectives where the incorporation of additional attenuation markers could contribute to develop safe rTCRV-based vaccines against pathogenic mammarenaviruses. Full article