Inhibition of the type I interferon response by the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a formidable battle horse for the study of viral immunology, as well as viral persistence and associated diseases. Investigations with LCMV have uncovered basic mechanisms by which viruses avoid elimination by the host adaptive immune response. In this study we show that LCMV also disables the host innate defense by interfering with beta interferon (IFN-beta) production in response to different stimuli, including infection with Sendai virus and liposome-mediated DNA transfection. Inhibition of IFN production in LCMV-infected cells was caused by an early block in the IFN regulatory factor 3 (IRF-3) activation pathway. This defect was restored in cells cured of LCMV, indicating that one or more LCMV products are responsible for the inhibition of IRF-3 activation. Using expression plasmids encoding individual LCMV proteins, we found that expression of the LCMV nucleoprotein (NP) was sufficient to inhibit both IFN production and nuclear translocation of IRF-3. To our knowledge, this is the first evidence of an IFN-counteracting viral protein in the Arenaviridae family. Inhibition of IFN production by the arenavirus NP is likely to be a determinant of virulence in vivo.     

RNA interference-mediated virus clearance from cells both acutely and chronically infected with the prototypic arenavirus lymphocytic choriomeningitis virus

Several arenaviruses, including Lassa fever virus, cause severe, often lethal hemorrhagic fever in humans. No licensed vaccines are available in the United States, and currently there is no efficacious therapy to treat this viral infection. Therefore the importance of developing effective antiviral approaches to combat pathogenic arenaviruses is clear. Moreover, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important model for the study of viral persistence and associated diseases, as well as for exploring therapies to treat viral chronic infections. The use of small interfering RNAs (siRNAs) to downregulate gene expression via RNA interference (RNAi) has emerged as a powerful genetic tool for the study of gene function. In addition, the successful use of siRNAs to target a variety of animal viruses has led us to consider RNAi as a potential novel antiviral strategy. We have investigated the use of RNAi therapy against LCMV. Here, we show that siRNAs targeting sequences within the viral L polymerase and Z mRNAs inhibit LCMV multiplication in cultured cells. Unexpectedly, the antiviral efficacy of RNAi-based therapy against LCMV was highly dependent on the method used to deliver effector siRNA molecules. Thus, transfection of chemically synthesized siRNA pools to L and Z was ineffective in preventing virus multiplication. In contrast, targeting of the same viral L and Z gene products with siRNAs produced inside cells using a replication-deficient recombinant adenovirus expression system inhibited LCMV multiplication very efficiently. Notably, transduction with the replication-deficient recombinant adenovirus expression system to Z and L effectively cured persistently LCMV-infected cells, suggesting the feasibility of using RNAi therapy to combat viral chronic infections by riboviruses.     

Genetic and biochemical evidence for an oligomeric structure of the functional L polymerase of the prototypic arenavirus lymphocytic choriomeningitis virus

The arenavirus L protein has the characteristic sequence motifs conserved among the RNA-dependent RNA polymerase L proteins of negative-strand (NS) RNA viruses. Studies based on the use of reverse-genetics approaches have provided direct experimental evidence of the key role played by the arenavirus L protein in viral-RNA synthesis. Sequence alignment shows six conserved domains among L proteins of NS RNA viruses. The proposed polymerase module of L is located within its domain III, which contains highly conserved amino acids within motifs designated A and C. We have examined the role of these conserved residues in the polymerase activity of the L protein of the prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), in vivo using a minigenome rescue assay. We show here that the presence of sequence SDD, a characteristic of motif C of segmented NS RNA viruses, as well as the presence of the highly conserved D residue within motif A of L proteins, is strictly required for the polymerase activity of the LCMV L protein. The strong dominant negative phenotype associated with many of the mutants examined and results from coimmunoprecipitation studies provided genetic and biochemical evidence, respectively, for the requirement of the L-L interaction for the polymerase activity of the LCMV L protein.     

Characterization of ribonucleoproteins and ribosomes isolated from lymphocytic choriomeningitis virus.

Disruption of purified lymphocytic choriomeningitis (LCM) virus with Nonidet P-40 in 0.5 M KCl followed by sucrose gradient centrifugation in 0.3 M KCl led to the isolation of two viral nucleoproteins (RNPs) as well as 40S and 60S ribosomal subunits. The largest viral RNP sedimented heterogenously at 123S to 148S and was associated with 23S and 31S viral RNA. The other viral RNP sedimented at 83S and was associated with 23S viral RNA. The buoyant density in CsCl was determined to be 1.32 g/cm3 for the viral RNP. Densities of 1.52 and 1.60 g/cm3 were determined for the 40S and 60S subunits, similar to those of the BHK-21 cells subunits dissociated by 0.5 M KCl. The viral RNPs were partly sensitive to RNase.     

Cellular entry of lymphocytic choriomeningitis virus

In contrast to most enveloped viruses that enter the host cell via clathrin-dependent endocytosis, the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) enters cells via noncoated vesicles that deliver the virus to endosomes, where pH-dependent membrane fusion occurs. Here, we investigated the initial steps of LCMV infection. We found that the attachment of LCMV to its cellular receptor α-dystroglycan occurs rapidly and is not dependent on membrane cholesterol. However, subsequent virus internalization is sensitive to cholesterol depletion, indicating the involvement of a cholesterol-dependent pathway. We provide evidence that LCMV entry involves an endocytotic pathway that is independent of clathrin and caveolin and that does not require the GTPase dynamin. In addition, neither the structural integrity nor the dynamics of the actin cytoskeleton are required for infection. These findings indicate that the prototypic Old World arenavirus LCMV uses a mechanism of entry that is different from clathrin-mediated endocytosis, which is used by the New World arenavirus Junin virus, and pathways used by other enveloped viruses.     

A-to-G hypermutation in the genome of lymphocytic choriomeningitis virus

The interferon-inducible adenosine deaminase that acts on double-stranded RNA (ADAR1-L) has been proposed to be one of the antiviral effector proteins within the complex innate immune response. Here, the potential role of ADAR1-L in the innate immune response to lymphocytic choriomeningitis virus (LCMV), a widely used virus model, was studied. Infection with LCMV clearly upregulated ADAR1-L expression and activity. The editing activity of ADAR1-L on an RNA substrate was not inhibited by LCMV replication. Accordingly, an adenosine-to-guanosine (A-to-G) and uracil-to-cytidine (U-to-C) hypermutation pattern was found in the LCMV genomic RNA in infected cell lines and in mice. In addition, two hypermutated clones with a high level of A-to-G or U-to-C mutations within a short stretch of the viral genome were isolated. Analysis of the functionality of viral glycoprotein revealed that A-to-G- and U-to-C-mutated LCMV genomes coded for nonfunctional glycoprotein at a surprisingly high frequency. Approximately half the GP clones with an amino acid mutation lacked functionality. These results suggest that ADAR1-L-induced mutations in the viral RNA lead to a loss of viral protein function and reduced viral infectivity. This study therefore provides strong support for the contribution of ADAR1-L to the innate antiviral immune response.     

Long-lived signal peptide of lymphocytic choriomeningitis virus glycoprotein pGP-C

Signal peptides (SPs) direct nascent secretory and membrane proteins to the membrane of the endoplasmic reticulum. They are usually cleaved from the nascent polypeptide by signal peptidase and then further proteolytically processed. The SP of the pre-glycoprotein (pGP-C) of the lymphocytic choriomeningitis virus SPGP-C (signal peptide of pGP-C) shows different properties: 1) The SPGP-C is unusually long (58 amino acid residues) and contains two hydrophobic segments interrupted by a lysine residue. 2) The SPGP-C is cleaved only from a subset of pGP-C proteins. A substantial portion of pGP-C accumulates that still contains the SPGP-C.3)The cleaved SPGP-C is rather long-lived (t(1/2) of more than 6 h). 4) The cleaved SPGP-C resides in the membrane and is resistant to digestion with proteinase K even in the presence of detergents, suggesting a very compact structure. 5) SPGP-C accumulates in virus particles. These unusual features of the cleaved SPGP-C suggest that SPGP-C not only targets the nascent pGP-C to the endoplasmic reticulum membrane but also has additional functions in lymphocytic choriomeningitis virus life cycle.     

Reactive oxygen species delay control of lymphocytic choriomeningitis virus

Cluster of differentiation (CD)8⁺ T cells are like a double edged sword during chronic viral infections because they not only promote virus elimination but also induce virus-mediated immunopathology. Elevated levels of reactive oxygen species (ROS) have been reported during virus infections. However, the role of ROS in T-cell-mediated immunopathology remains unclear. Here we used the murine lymphocytic choriomeningitis virus to explore the role of ROS during the processes of virus elimination and induction of immunopathology. We found that virus infection led to elevated levels of ROS producing granulocytes and macrophages in virus-infected liver and spleen tissues that were triggered by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Lack of the regulatory subunit p47phox of the NADPH oxidase diminished ROS production in these cells. While CD8⁺ T cells exhibited ROS production that was independent of NADPH oxidase expression, survival and T-cell function was elevated in p47phox-deficient (Ncf1^−/−) mice. In the absence of p47phox, enhanced T-cell immunity promoted virus elimination and blunted corresponding immunopathology. In conclusion, we find that NADPH-mediated production of ROS critically impairs the immune response, impacting elimination of virus and outcome of liver cell damage.     

Contamination of transplantable murine tumors with lymphocytic choriomeningitis virus

Lymphocytic choriomeningitis virus (LCMV) was isolated from a transplantable tumor after mice bearing the tumor began to die prematurely. Tumor lines, mice and laboratory personnel that had an association with the index laboratory were tested for LCMV infection. Testing of tumor lines from the index laboratory and four other laboratories revealed that 16 of 55 tumor samples used in vivo and one of eight tumor samples maintained in vitro were contaminated with LCMV. Laboratory personnel and uninoculated mice that were exposed to infected tumors had no LCMV antibody. The use of carefully monitored seed stocks is recommended to protect transplantable tumors that may be inadvertently contaminated by viruses.