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Mammarenaviruses are enveloped viruses with a bisegmented negative-stranded RNA genome that encodes the nucleocapsid protein (NP), the envelope glycoprotein precursor (GPC), the RNA polymerase (L), and a RING matrix protein (Z). Viral proteins are synthesized from subgenomic mRNAs bearing a capped 5' untranslated region (UTR) and lacking 3' poly(A) tail. We analyzed the translation strategy of Tacaribe virus (TCRV), a prototype of the New World mammarenaviruses. A virus-like transcript that carries a reporter gene in place of the NP open reading frame and transcripts bearing modified 5' and/or 3' UTR were evaluated in a cell-based translation assay. We found that the presence of the cap structure at the 5' end dramatically increases translation efficiency and that the viral 5' UTR comprises stimulatory signals while the 3' UTR,specifically the presence of a terminal C+G-rich sequence and/or a stem-loop structure, down-modulates translation. Additionally, translation was profoundly reduced in eukaryotic initiation factor (eIF) 4G-inactivated cells, whereas depletion of intracellular levels of eIF4E had less impact on virus-like mRNA translation than on a cell-like transcript. Translation efficiency was independent of NP expression or TCRV infection. Our results indicate that TCRV mRNAs are translated using a cap-dependent mechanism, whose efficiency relies on the interplay between stimulatory signals in the 5' UTR and a negative modulatory element in the 3' UTR. The low dependence on eIF4E suggests that viral mRNAs may engage yet-unknown noncanonical host factors for a cap-dependent initiation mechanism. IMPORTANCE Several members of the Arenaviridae family cause serious hemorrhagic fevers in humans. In the present report, we describe the mechanism by which Tacaribe virus, a prototypic nonpathogenic New World mammarenavirus, regulates viral mRNA translation. Our results highlight the impact of untranslated sequences and key host translation factors on this process. We propose a model that explains how viral mRNAs outcompete cellular mRNAs for the translation machinery. A better understanding of the mechanism of translation regulation of this virus can provide the bases for the rational design of new antiviral tools directed to pathogenic arenaviruses., (Copyright © 2017 American Society for Microbiology.)

Tacaribe virus (TACV) is an arenavirus that is genetically and antigenically closely related to Junin virus (JUNV), the aetiological agent of Argentine haemorrhagic fever (AHF). It is well established that TACV protects experimental animals fully against an otherwise lethal challenge with JUNV. To gain information on the nature of the antigens involved in cross-protection, recombinant vaccinia viruses were constructed that express the glycoprotein precursor (VV-GTac) or the nucleocapsid protein (VV-N) of TACV. TACV proteins expressed by vaccinia virus were indistinguishable from authentic virus proteins by gel electrophoresis. Guinea pigs inoculated with VV-GTac or VV-N elicited antibodies that immunoprecipitated authentic TACV proteins. Antibodies generated by VV-GTac neutralized TACV infectivity. Levels of antibodies after priming and boosting with recombinant vaccinia virus were comparable to those elicited in TACV infection. To evaluate the ability of recombinant vaccinia virus to protect against experimental AHF, guinea pigs were challenged with lethal doses of JUNV. Fifty per cent of the animals immunized with VV-GTac survived, whereas all animals inoculated with VV-N or vaccinia virus died. Having established that the heterologous glycoprotein protects against JUNV challenge, a recombinant vaccinia virus was constructed that expresses JUNV glycoprotein precursor (VV-GJun). The size and reactivity to monoclonal antibodies of the vaccinia virus-expressed and authentic JUNV glycoproteins were indistinguishable. Seventy-two per cent of the animals inoculated with two doses of VV-GJun survived lethal JUNV challenge. Protection with either VV-GJun or VV-GTac occurred in the presence of low or undetectable levels of neutralizing antibodies to JUNV.

Experimental infection of three mouse strains with a non-pathogenic mutant of Junin virus named Cl67 was compared with respect to the parental XJCl3 strain. After intracerebral (ic) or intraperitoneal inoculation, XJCl3 was highly virulent for 2 day-old C3H/HeJ, OF1, and BALB/cJ mouse strains, whereas its derivative Cl67 was attenuated. Survival of the Cl67-infected mouse was associated with a restricted replication at the site of inoculation which would impair spread of virus. Thus, the reduced virulence of Cl67 for suckling mice is independent of the mouse strain and the route of viral entry. When Cl67 was preinoculated ic 10 days before the challenge inoculation with XJCl3 by the same route, mice were partially protected from lethal infection. Since neutralizing antibodies were first detected at 30 days post-infection, an interference mechanism is postulated as a mechanism of protection of the mice.

The growth characteristics, cytopathogenicity and viral polypeptides of the virulent strain XJ of Junin virus (JV), its attenuated derivative XJC13 and another naturally attenuated JV strain, IV4454, were comparatively studied. IV4454 and XJC13 viruses showed the highest and lowest cytopathology for Vero cells, respectively, as measured by plaque morphology, cell viability and inhibition of host cell protein synthesis. The kinetics and electrophoretic patterns of viral polypeptides in infected cell extracts were very similar among the three strains, whereas differences were detected in the surface glycoprotein GP38 by peptide mapping after limited proteolysis.

The XJC13 strain of Junin virus (JV) and the mouse-attenuated mutant C167 showed different GP38 peptide mapping after limited proteolysis with ficin and papain; viral infectivity of both viruses also exhibited a different susceptibility to protease treatment. A correlation between envelope glycoprotein alteration and JV virulence in neonatal mice is proposed.

C167, a mutant derived from the XJC13 strain of Junin virus, is highly attenuated in its pathogenic properties for newborn mice. Whereas 10(2).PFU of XJC13 injected intracerebrally killed 100% of two-day-old mice, the mutant showed no detectable lethality. Survival of mice infected with C167 was associated with a reduced and delayed virus replication in brain and a defective spread of virus from the site of inoculation to the other tissues, including spleen, kidney, thymus, liver, peritoneal cells and serum. As an apparent consequence of the restricted replication of C167 in mice, no detectable interferon induction and low levels of neutralizing antibodies were observed. Analysis of multiplication kinetics of C167 and XJC13 in different cell cultures in vitro has confirmed that the attenuated phenotype of C167 was related to a specific inefficient replication in murine cells. This host-range restriction was due to a combination of adsorption and penetration blockage.

The virulence in neonatal mice of a temperature-sensitive mutant of Junin virus, named C167, was studied. The thermosensitive properties of this mutant were tested by titration on Vero cells at 37 and 40 degrees C. The ratio of infectivity 40/37 was approximately 100-fold lower for C167 with respect to XJC13 (Table 1). The attenuation of C167 was determined by measurement of mean survival time and 50% lethal dose after intracerebral injection of 2 and 11 day old mice. For C167 the lethality index (expressed ad the ratio TCID50/LD50) was greater than 580, while for XJC13 the index was 4.4 (Table 2). The lack of virulence of C167 was correlated with a restricted ability to replicate in suckling mouse brains. By contrast, the mutant and the parental virus grew to a similar titre in Vero cells (Figure 2). Both viruses were indistinguishable in cross-neutralization tests using hyperimmune antisera.