PubMed · 206025
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S Ia Gaĭdamovich, M Iu Kocherovskaia. [Arenaviruses].. https://pubmed.ncbi.nlm.nih.gov/206025/
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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-alpha 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-alpha gene expression and even down-regulates LPS-stimulated TNF-alpha 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-alpha and IL-8 expression could explain the absence of inflammatory and effective immune responses in severe cases of Lassa HF.
The development of safe and effective antiviral agents has been a slow process, largely because of the difficulty in distinguishing between virus and host functions; materials toxic to the virus are frequently harmful also to the host in which the agent resides. Recently, techniques which target nucleic acid sequences as a means of reducing gene expression have emerged. This antisense armamentarium includes ribozymes, RNA enzymes which cleave other RNA molecules in a sequence-specific manner. We wish to assess the ability of ribozymes to control animal virus infection. Reasoning that the viruses most vulnerable to ribozyme intervention will be those whose complete life cycle is based on RNA (with no DNA stage), we have begun to develop ribozymes directed toward lymphocytic choriomeningitis virus (LCMV), the prototype of the arenavirus family. Using ribozymes of the hammerhead variety, we have identified several sites on the LCMV genome which can be efficiently cleaved in trans. The efficiency of cleavage is site dependent, and we demonstrate that secondary structure at the target site can abolish ribozyme cleavage. Computer-assisted analysis indicates that much of the LCMV genome may be involved in base pairing, which may render it similarly resistant to ribozyme attack. The few remaining open regions of LCMV lack a GUC target site, on which most studies to date have relied. Here we show that AUC, CUC, and AUU are alternative sites which can be cleaved by trans-acting ribozymes. This finding is important given the aforementioned restriction of available sites, imposed by secondary structure.
A Tacaribe virus in vitro system for RNA synthesis was established and found in large part to faithfully reproduce RNA synthesis in vivo. Similar to influenza virus and bunyavirus in vitro systems, this system was also highly dependent on added oligonucleotides. Of the eight tested, only three were active, in the order GpC greater than CpG greater than ApApC. Determination of the 5' ends of the transcripts suggested that the oligonucleotides were acting as primers. In particular, whereas stimulation with CpG (complementary to positions +1 and +2 of the template) led to RNAs whose 5' ends were at position +1 as expected, GpC stimulation led to transcripts whose 5' ends were at position -1 rather than at position +2, as GpC is complementary to positions +2 and +3 of the template. This finding suggests a model for the initiation of genome replication in which pppGpC is first made on the template at positions +2 and +3 but slips backwards on the template so that the 5' end is at position -1 before elongation can continue.