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.

Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike.

Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, which is composed of two viral glycoprotein subunits, GP1 and GP2, and the stable signal peptide. Of these, GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis. The molecular structure of the spike and conformational rearrangements induced by low pH, prior to fusion, remain poorly understood. Here, we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography. Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution. The spikes are trimeric, cover the virion envelope, and connect to the underlying matrix. Structural changes to the spike, following acidification, support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits.

Characterization of the Lassa virus matrix protein Z: electron microscopic study of virus-like particles and interaction with the nucleoprotein (NP).

Lassa virus is the causative agent of a hemorrhagic fever endemic in west Africa. The RNA genome of Lassa virus encodes the glycoprotein precursor GP-C, a nucleoprotein (NP), the viral polymerase L and a small protein Z (11 kDa). Here, we analyze the role of Z protein for virus maturation. We have recently shown that expression of Z protein in the absence of other viral proteins is sufficient for the release of enveloped Z-containing particles. In this study, we examined particles secreted into the supernatant of a stably Z protein-expressing CHO cell line by electron microscopy. The observed Z-induced virus-like particles did not significantly differ in their morphology and size from Lassa virus particles. Mutation of two proline-rich domains within Z which are known to drastically reduce the release of virus-like particles, had no effect on the cellular localization of the protein nor on its membrane-association. Furthermore, we present evidence that Z interacts with the NP. We assume that Z recruits NP to cellular membranes where virus assembly takes place. We conclude from our data that Lassa virus Z protein plays an essential role in Lassa virus maturation.

Expression of the Lassa virus nucleocapsid protein in insect cells infected with a recombinant baculovirus: application to diagnostic assays for Lassa virus infection.

The coding region of the gene for the nucleocapsid protein of Lassa virus has been inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) using the transfer vector pAcYM1, so that expression of the foreign DNA is under the control of the promoter of the AcNPV polyhedrin gene. Infection of cultured Spodoptera frugiperda cells with recombinant virus resulted in the synthesis of high levels of a protein that was indistinguishable from the authentic Lassa virus protein by SDS gel electrophoresis and immunoblotting with a variety of specific immune sera and monoclonal antibodies (MAbs). The kinetics of appearance of the protein were comparable to those of polyhedrin production in wild-type AcNPV-infected cells. The recombinant material was antigenic when used in ELISA for Lassa virus-specific antibodies, reacting well with MAbs specific for the nucleocapsid protein and with sera from experimentally infected guinea-pigs. The recombinant ELISA was able to clearly distinguish sera from human cases of Lassa fever against a panel of known negative sera of African origin. Recombinant-infected insect cells were an effective substitute for mammalian cells infected with Lassa virus itself in the immunofluorescence assay for Lassa virus-specific antibodies. This system offers attractive alternatives to the use of Lassa virus-infected materials as reagents in diagnostic procedures.

Preparation and biochemical characterization of Lassa virus subvirion fractions.

14C-labelled Lassa virus was purified by isopycnic centrifugation and split to subvirion fractions. The purified virus was treated with nonionic detergents Nonidet P-40 (NP-40) and with octylglycoside. After ultracentrifugation in urographin density gradient, two subvirion fractions with buoyant density of 1.24-1.26 and 1.08-1.10 g/cm3 were obtained. The first fraction corresponded to the nucleocapsid of Lassa virus: it contained a protein with molecular mass of 60 kDa, the L and S segments of the genomic RNA. The second one contained a protein with molecular mass of 48 kDa and represented, apparently, envelope fraction of virus particles.

[Electron microscopic study of Lassa virus]. / Elektronno-mikroskopicheskoe izuchenie virusa Lassa.

Ultrafiltration through hollow fibrous filters followed by purification in interrupted and linear urografin gradients yielded a Lassa virus suspension of high concentration. The use of gamma-irradiation for inactivation of the frozen virus suspension (-70 degrees C) caused no apparent structural changes of virions and made it possible to examine Lassa virus in electron microscope by negative staining. The observed virus particles in their morphology and sizes did not differ from previously described particles of other members of the Arenaviridae family. In ultrathin sections of Lassa virus-infected Vero cells, atypical virions were sometimes visible alongside with typical particles. Within one type of such particles no ribosome-like granules could be detected. Such "hollow" particles may possibly be defective virions. Another kind of atypical particles contained homogeneous electron-dense core and resembled mycoplasma. Of greatest interest are the particles with heterogeneous core in which "sandy" granules can be distinguished. The presence of greater amounts of uranophilic material than usually may be explained by getting into the virion in the process of its formation of a greater amount of genetic material than that present in typical virions.

[Recent Lassa, Marbourg and Ebola viruses in African tropical viruses. I. Semiology--physiopathology--diagnosis--treatment (author's transl)]. / Les virus Lassa, Marbourg et Ebola, nouveaux venus en pathologie tropicale africaine. I. Sémiologie--Physio-pathologie--Diagnostic--Traitement.

Three new viruses have been identified in Africa during the present decade. They may cause sporadic cases or limited outbreaks, and they are probably endemic in areas which are still ill-defined. Severe forms of infection lead to the haemorrhagic syndrome or to hypovolemic shock, the physiopathology of which is being studied. The case-fatality ratio of severe cases is between 30 and 85 per cent. Nosocomial outbreaks have been observed, but they can be avoided if appropriate barrier nursing measures are carried out for the treatment of patients or adequate protection measures for sampling and examination of laboratory specimens. As such cases may be transferred outside the endemic zone, this implies that countries receiving travellers from Africa should have hospitals with specialized units for strict isolation and treatment of these patients.

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.

The pathology of human Lassa fever.

Pathological findings have been described in only a small number of cases of Lassa fever since the virus was first isolated in 1969. Morphologically, eosinophilic necrosis of hepatocytes was the most frequent finding and focal necroses, often extensive, were present in most cases. These findings are similar to the lesions previously described in Argentinian and Bolivian haemorrhagic fever. Focal interstitial pneumonitis, focal tubular necrosis in the kidney, lymphocytic infiltration of the splenic veins, and partial replacement of the splenic follicles by amorphous eosinophilic material have been described, but the significance of these findings is unclear. More detailed and sophisticated investigations are required in the future if pathogenetic mechanisms are to be unravelled.