Structural insight into arenavirus replication machinery.

Arenaviruses can cause severe haemorrhagic fever and neurological diseases in humans and other animals, exemplified by Lassa mammarenavirus, Machupo mammarenavirus and lymphocytic choriomeningitis virus, posing great threats to public health1-4. These viruses encode a large multi-domain RNA-dependent RNA polymerase for transcription and replication of the viral genome5. Viral polymerases are one of the leading antiviral therapeutic targets. However, the structure of arenavirus polymerase is not yet known. Here we report the near-atomic resolution structures of Lassa and Machupo virus polymerases in both apo and promoter-bound forms. These structures display a similar overall architecture to influenza virus and bunyavirus polymerases but possess unique local features, including an arenavirus-specific insertion domain that regulates the polymerase activity. Notably, the ordered active site of arenavirus polymerase is inherently switched on, without the requirement for allosteric activation by 5'-viral RNA, which is a necessity for both influenza virus and bunyavirus polymerases6,7. Moreover, dimerization could facilitate the polymerase activity. These findings advance our understanding of the mechanism of arenavirus replication and provide an important basis for developing antiviral therapeutics.

Description of Guanarito virus (Arenaviridae: Arenavirus), the etiologic agent of Venezuelan hemorrhagic fever.

This paper characterizes Guanarito virus, the etiologic agent of Venezuelan hemorrhagic fever. Based on its morphology and antigenic properties, Guanarito virus appears to be a new member of the Tacaribe complex of the genus Arenavirus, family Arenaviridae. Complement fixation and indirect fluorescent antibody tests showed that Guanarito virus and its antiserum are broadly cross-reactive with other members of the Tacaribe complex, but it can be differentiated from other members of the complex by neutralization test. Guanarito virus causes mortality in suckling mice and adult guinea pigs, but not in adult mice. Inoculated rhesus monkeys developed viremia and became ill; however, they subsequently recovered and responded with production of antibody. To date, all isolates of Guanarito virus have come from sick persons or wild rodents living within a single geographic focus in the central plains of Venezuela.

Replication and physical parameters important for preparing purified Junin virus.

Junin virus (JV) is an Arenavirus and the causative agent of Argentine hemorrhagic fever (AHF), an often fatal human disease. The attenuated strain XJ-clone 3 (XJC13) of JV, after being tested in humans, has been considered a promising vaccine. We found that synthesis of JV XJC13 reaches a peak 2 days after infection and the kinetics of synthesis are little affected by the multiplicity of infection (MOI) in a range from 0.125 to 1.00. Virus synthesis is sensitive to actinomycin D, indicating that cellular biosynthesis is required for viral replication. Combined precipitation and ultracentrifugation of supernatant from virus-infected cells yielded large amounts of concentrated and purified virion that banded in sucrose as a single peak with average density 1.177 +/- 0.015 g/ml. Purified virions have an average diameter of 203 +/- 23 nm by electron microscopy and an average sedimentation coefficient of 454 +/- 27 S. The results from the present study should assist in the preparation of large amounts of attenuated Junin virus which are required for vaccination against and diagnosis of Argentine hemorrhagic fever.

Immunoperoxidase tracing of Junin virus neural route after footpad inoculation.

To determine the pathway adopted by peripherally inoculated Junin virus (JV) to reach the CNS, rat tissues were serially harvested to trace the sequence of viral progression from right hind footpad to brain. Immunoperoxidase (PAP) labeling of viral antigen, concomitantly with infectivity assays and histological examination of each selected sample, were carried out. As from the 2nd week post-infection (pi), neurological disease inducing 100% mortality at 1 month was evident. At day 5 pi, viral antigen was first detected at footpad level in epidermic and dermic cells, as well as in neighbouring myocytes; labeled macrophages infiltrating small nerve branches were also disclosed. As from 10-15 days pi, viral antigen became apparent along ipsilateral sciatic nerve structures and within lumbar spinal ganglion neurons, followed by a fast viral spread throughout CNS neurons that involved spinal cord and brain. Concurrent histopathology featured minimal inflammatory reaction together with generalized astrocytic activation. Hematogenous viral transport was negligible, since JV was isolated much earlier and in higher infectivity titers in neural tissues than in blood. It may be concluded that after viral replication in footpad, JV neural route was demonstrated by its PAP labeling from peripheral nerves to cerebral cortex.

Structural components of the arenavirus Pichinde.

Purified Pichinde virions grown in monolayers of BHK-21 cells were found to contain three major species of virion proteins as described previously (Ramos et al., J. Virol. 10:661-667, 1972). Two of the proteins were glycosylated (G1, molecular weight = 64,000; G2, molecular weight = 38,000) and were present in similar proportions on the outer surface of the virions. A third protein (N, molecular weight = 66,000) was not glycosylated and, in association with the viral RNA species, was the major protein component of the viral nucleocapsids. An estimate of the approximate number of molecules of these three major proteins per virion was made. Minor amounts of other proteins were also routinely observed in Pichinde virus preparations. None of the three major protein species were phosphorylated to any significant exten, nor did they contain sulfated components. Two virion RNA species (L and S), but no 18S rRNA species, were detected in Pichinde virus preparations obtained from infected BHK-21 cells.

The circular, segmented nucleocapsid of an arenavirus-Tacaribe virus.

The nucleocapsid structures of Tacaribe virus, a member of the Arenaviridae, were purified from detergent-treated virus particles by equilibrium density gradient centrifugation. Negative-contrast electron microscopy indicated that they were coiled, circular filaments. They had a mean diam. of 5 to 10 nm and two predominant length classes of 640 nm and 1300 nm were found.

Junin virus structural proteins.

Polyacrylamide gel electrophoresis of purified Junin virus revealed six distinct structural polypeptides, two major and four minor ones. Four of these polypeptides appeared to be covalently linked with carbohydrate. The molecular weights of the six proteins, estimated by coelectrophoresis with marker proteins, ranged from 25,000 to 91,000. One of the two major components (number 3) was identified as a nucleoprotein and had a molecular weight of 64,000. It was the most prominent protein and was nonglycosylated. The other major protein (number 5), with a molecular weight of 38,000, was a glucoprotein and a component of the viral envelope. The location on the virion of three additional glycopeptides with molecular weights of 91,000, 72,000, and 52,000, together with a protein with a molecular weight of 25,000, was not well defined.

Ultrastructural and immunohistochemical studies in five cases of Argentine hemorrhagic fever.

Ultrastructural and immunohistochemical studies on tissues from five patients with Argentine hemorrhagic fever revealed previously undetected lesions caused by the viral infection. Two types of particle were seen in the cells of all organs examined. The particles had some characteristics similar to those described for arenaviruses. However, the virus-like particles were intracellular, had a single membrane, and apparently originated by a process of budding into the endoplasmic reticulum cisternae. Intranuclear bodies and three types of cytopolasmic change were observed in conjunction with the virus-like particles; Antigenic determinants of Junin virus were demonstrated in cells of all organs examined. Immunohistochemical experiments also indicated alterations in the cellular mechanisms of protein synthesis. Until now the pathogenesis of human diseases produced by arenaviruses has not been established. The results of this study suggest that in Argentine hemorrhagic fever the virus is responsible for a direct pathogenic action.

Morphology and morphogenesis of arenaviruses.

Arenaviruses have unique structural characteristics; they are pleomorphic, have a mean diameter of 110-130 nm, and consist of a membranous envelope with surface projections surrounding an interior containing ribosomes and filaments. Virus particles bud from plasma membranes of infected cells and in many cases large intracytoplasmic inclusion bodies are formed. These characteristics allow generic identification, but not differentiation of individual viruses. Ultrastructural identification of virus particles and pathological processes in infected tissues of man and experimental animals is important in understanding the nature of arenaviral pathogenesis Such identification also contributes to our understanding of the mechanisms of viral shedding and transmission in reservoir host species.