Maria S. Salvato

Lassa and Mopeia Virus Replication in Human Monocytes/Macrophages and in Endothelial Cells: Different Effects on IL-8 and TNF-α Gene Expression

Cells of the mononuclear and endothelial lineages are targets for viruses which cause hemorrhagic fevers (HF) such as the filoviruses Marburg and Ebola, and the arenaviruses Lassa and Junin. A recent model of Marburg HF pathogenesis proposes that virus directly causes endothelial cell damage and macrophage release of TNF‐α which increases the permeability of endothelial monolayers [Feldmann et al., 1996]. We show that Lassa virus replicates in human monocytes/macrophages and endothelial cells without damaging them. Human endothelial cells (HUVEC) are highly susceptible to infection by both Lassa and Mopeia (a non‐pathogenic Lassa‐related arenavirus). Whereas monocytes must differentiate into macrophages before supporting even low level production of these viruses, the virus yields in the culture medium of infected HUVEC cells reach more than 7 log10 PFU/ml without cellular damage. In contrast to filovirus, Lassa virus replication in monocytes/macrophages fails to stimulate TNF‐α gene expression and even down‐regulates LPS‐stimulated TNF‐α mRNA synthesis. The expression of IL‐8, a prototypic proinflammatory CXC chemokine, was also suppressed in Lassa virus infected monocytes/macrophages and HUVEC on both the protein and mRNA levels. This contrasts with Mopeia virus infection of HUVEC in which neither IL‐8 mRNA nor protein are reduced. The cumulative down‐regulation of TNF‐α and IL‐8 expression could explain the absence of inflammatory and effective immune responses in severe cases of Lassa HF. J. Med. Virol. 59:552–560, 1999.

Biochemical and immunological evidence that the 11 kDa zinc-binding protein of lymphocytic choriomeningitis virus is a structural component of the virus.

The completed sequence of the arenavirus, lymphocytic choriomeningitis virus, revealed a new gene encoding a small protein with a single zinc-binding domain. The cDNA for this gene has been expressed in E. coli to produce fusion protein that has been used to raise antisera. The antisera facilitated the positive identification of the p11 Z gene product as a structural component of the virion. A related arenavirus, Tacaribe, has a comparable p11 gene product. The abundance of the p11 Z protein relative to other virion components has been determined by metabolic labeling. Triton X-114 extraction and dimethyl suberimidate-HCl crosslinking indicate that the p11 Z protein is a hydrophobic protein associated with the nucleocapsid of the virion core.

The promyelocytic leukemia protein PML has a pro-apoptotic activity mediated through its RING domain.

The promyelocytic leukemia protein PML is known to form nuclear multiprotein complexes which are compromised in several pathogenic conditions including acute promyelocytic leukemia. We show that in cells infected with a single stranded RNA virus, which relocates PML bodies to the cytoplasm, the infected cells are more resistant to serum starvation induced apoptosis than their uninfected counterparts. Antisense PML oligonucleotides increase cell survival under serum deprivation conditions indicating that PML is directly involved in the apoptotic activity. Transient transfection studies have indicated that this pro‐apoptotic activity of PML is mediated through the zinc binding region known as the RING finger. Viral attack of PML nuclear bodies appears to allow the virus to deregulate host cell apoptotic machinery in order to establish chronic infection.

Molecular Biology of the Prototype Arenavirus, Lymphocytic Choriomeningitis Virus

Structural analysis of a well-characterized virus such as lymphocytic choriomeningitis virus (LCMV) is of particular importance in interpret-ing complex biological phenomena. Complete determination of the nu-cleotide sequence of LCMV establishes the structural features and cod-ing capacity of the virus genome. It enables further studies on the gene expression and replication mechanisms of arenaviruses, with potential significance for our understanding of viral persistence and pathogenesis.

Murine immune responses to mucosally delivered Salmonella expressing Lassa fever virus nucleoprotein

Arenaviruses are emerging pathogens known to infect via the mucosa, however no formal attempts to make mucosal vaccines have been undertaken. Here we describe a recombinant aroA attenuated Salmonella typhimurium that expresses the nucleoprotein (NP) gene of Lassa fever virus (LAS). The complete NP gene was cloned downstream of the bacterial groEL promotor and integrated into the aroA locus of S. typhimurium. Lassa NP protein was detected in whole cell extracts from the recombinant Salmonella by immunoblot analysis with serum from Lassa-infected people. Mice were inoculated by intragastric intubation with 5 x 10(9) S. typhimurium and boosted with the same recombinant Salmonella 21 days after the primary inoculation. Both local mucosal IgA and serum immunoglobulins against Lassa NP were observed. Splenic cytotoxic T-lymphocyte responses to LAS NP were detected after the boost and they cross-reacted with target cells infected with the related arenavirus, lymphocytic choriomeningitis virus. Recombinant Salmonella elicits humoral and cell mediated immune responses against Lassa fever virus in mice and should be considered as a potential vaccine strategy in man.

Sequence Comparison of the Large Genomic RNA Segments of Two Strains of Lymphocytic Choriomeningitis Virus Differing in Pathogenic Potential for Guinea Pigs

Two strains of lymphocytic choriomeningitis virus (LCMV) differ in their ability to cause a lethal disease in outbred guinea pigs: the Armstrong (ARM) strain is not lethal at high doses (106 PFU), whereas the WE strain is lethal at less than 10 PFU inoculated intraperitoneally. The high pathogenic potential of LCMV WE has been mapped to the larger (L) of the two genomic RNA segments by genetic reassortment analysis (Riviere, Y., Ahmed, R., Southern, P.J., Buchmeier, M. J. and Oldstone, M. B. A., J. Virol. 55, 704–709, 1985). Here we describe the completed sequence of the LCMV WE L RNA, and its comparison to the L RNA of the non-virulent strain, LCMV ARM. Similar to the L RNA of LCMV ARM, the L RNA of WE is 7.2 kb long and contains two open reading frames (ORFs): the 5′ ORF encodes a small RING finger (zinc-binding) protein, p11 Z, and the 3′ ORF encodes the putative RNA-dependent RNA polymerase (RdRp or L protein). Comparison of nucleotide sequences for both viruses revealed 84% L RNA homology. At the amino acid level similarity between the two strains is 87% in the Z ORF, and 88% in the RdRp ORF. The most divergent regions are found in the N-terminal parts of the RdRp and Z proteins and are most likely to account for differences in pathogenic potential.

Vaccination protects β2 microglobulin deficient mice from immune mediated mortality but not from persisting viral infection

Intracranial (i.c.) infection of immunocompetent mice with lymphocytic choriomeningitis virus (LCMV) results in immunopathological lethal meningitis mediated by CD8+ cytotoxic T lymphocytes (CTL). Vaccination of immunocompetent mice elicits a CD8+ CTL response that can protect the mice from lethal meningitis. beta 2 microglobulin-deficient (beta 2m-/-) mice are deficient in CD8+ CTL, exhibit CD4+ CTL, and, after i.c. LCMV infection, undergo a less severe meningitis with decreased mortality and additionally develop a wasting disease. Both wasting disease and mortality in beta 2m-/- mice are mediated by CD4+ T cells. We studied the effects of vaccination and challenge dose on weight loss, mortality and viral clearance after i.c. LCMV infection in beta 2m-/- mice. Unvaccinated beta 2m-/- mice had significant weight loss and mortality at doses of 200 and 10(3) p.f.u. LCMV, while a dose of 10(6) p.f.u. LCMV elicited significant mortality but less weight loss. Vaccination with u.v.-inactivated LCMV in complete Freunds adjuvant or with vaccinia virus expressing the LCMV glycoprotein or nucleoprotein genes protected beta 2m-/- mice from mortality but not weight loss after 200 p.f.u. LCMV challenge. Although protected from mortality, beta 2m-/- mice were unable to clear LCMV from their brains or spleens. Therefore, we show that vaccination can protect against lethal immune-meningitis in the face of persistent infection.