Genetic mapping of lymphocytic choriomeningitis virus pathogenicity: virulence in guinea pigs is associated with the L RNA segment.

The Armstrong CA 1371 (ARM) and WE strains of lymphocytic choriomeningitis virus (LCMV) differ in the ability to produce disease in adult guinea pigs. Infection with the ARM strain is not lethal, even at high virus doses (greater than 10,000 PFU), whereas the WE strain causes 100% mortality even at low doses (less than 10 PFU). To determine the genetic basis of this virulence, intertypic reassortants were made between the ARM and WE strains of LCMV. The two reassortants with the genotypes WE/ARM (L segment of WE and S segment of ARM) and ARM/WE (L segment of ARM and S segment of WE) were tested for their pathogenicity in guinea pigs. The ARM/WE reassortant was avirulent like the ARM/ARM parental strain. Minimal viral replication was observed in organs of guinea pigs inoculated with 10(2) or 10(5) PFU of ARM/ARM or ARM/WE, and all animals survived. In contrast, the WE/ARM reassortant was highly virulent like the WE/WE parental strain and killed all of the infected animals. High levels of viral replication were observed in guinea pigs infected with the latter two strains. In contrast to these in vivo observations, both the parental strains and the ARM/WE or WE/ARM reassortants had similar growth potential in cultured guinea pig fibroblasts. Thus, the L RNA segment of LCMV WE is important for viral replication in vivo and is associated with fatal acute disease after infection of adult guinea pigs.     

Virus-induced immune complex disease: identification of specific viral antigens and antibodies deposited in complexes during chronic lymphocytic choriomeningitis virus infection.

Structural proteins of LCMV were identified and their role in the immune complex glomerulonephritis of LCMV carrier mice was examined. Purified LCMV contained three major polypeptides, a single nonglycosylated nucleoprotein with an estimated m.w. of 63,000, and two surface glycoproteins of 54,000 and 35,000. Deposition of nucleoprotein antigen in the glomeruli of LCMV carrier mice of several strains was demonstrated by immunofluorescent staining with a monospecific antibody. In addition, Ig eluted from kidneys of three strains of LCMV carrier mice was shown by immune precipitation to react against all of major viral polypeptides of LCMV. Antibody from normal mice, and from mice with immune complex disease unrelated to LCMV did not show deposition of LCMV antigen in glomeruli, and Ig eluted from the kidneys of these mice did not react against LCMV antigens. Hence, mice infected at birth with LCMV and persistently infected throughout their life make antibodies to all the known structural polypeptides of the virus.     

Recombinant expression of lymphocytic choriomeningitis virus strain WE glycoproteins: a single amino acid makes the difference

Cytoplasmic vector systems are generally used for expression of lymphocytic choriomeningitis virus (LCMV) proteins. However, we achieved high levels of cell surface glycoproteins using a standard nuclear expression plasmid. Expression was independent of other LCMV proteins but was blocked by a missense mutation within the original LCMV(WE) glycoprotein cDNA.     

Resistance of lymphocytic choriomeningitis virus to alpha/beta interferon and to gamma interferon.

The susceptibility to alpha/beta interferon (IFN-alpha/beta) or to gamma interferon (IFN-gamma) of various lymphocytic choriomeningitis virus (LCMV) strains was evaluated in C57BL/6 mice and in various cell lines. Anti-IFN-gamma treatment in vivo revealed that the LCMV strains Armstrong, Aggressive, and WE were most susceptible to IFN-gamma whereas Traub, Cl 13-Armstrong, and Docile were resistant. The same pattern of susceptibility to recombinant IFN-gamma was observed in vitro. In vivo treatment with anti-IFN-alpha/beta showed a sizeable increase in replication of Aggressive, Armstrong, and WE; effects were less pronounced for Docile, Cl 13-Armstrong, or Traub. Correspondingly, WE, Armstrong, and Aggressive were all relatively sensitive to purified IFN-alpha/beta in vitro, and Cl 13-Armstrong, Docile, and Traub were more resistant. Overall, there was a good correlation between the capacity of LCMV strains to establish a persistent infection in adult immunocompetent mice and their relative resistance to IFN-gamma and IFN-alpha/beta.     

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus. V. Recognition is restricted to gene products encoded by the viral S RNA segment

Lymphocytic choriomeningitis virus (LCMV) Armstrong (ARM) strain-specific, H-2d-restricted CTL effectively lyse syngeneic targets infected by LCMV ARM, but show reduced killing of LCMV Pasteur (PAST) strain-infected H-2d cells. We have reassorted the two RNA segments, large (L) and small (S), of LCMV ARM and PAST to generate LCMV with genotypes of L ARM/S PAST and L PAST/S ARM. By using these reassortants and both LCMV primary CTL and CTL clones, we report that the induction, recognition, and lysis of LCMV-specific CTL depend on the S RNA segment and the genes it encodes.     

Molecular basis of viral persistence: a single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with suppression of the antiviral cytotoxic T-lymphocyte response and establishment of persistence.

Isolates of lymphocytic choriomeningitis virus (LCMV) that elicit a cytotoxic T-lymphocyte response (CTL+) have been compared with isolates that suppress the CTL response (CTL-) in an effort to map this phenotype. A single amino acid change in the glycoprotein of the LCMV Armstrong (ARM) strain is consistently associated with the CTL- trait and the ability of the virus to persist (P+). The CTL+ P- parental strain spontaneously gives rise to CTL- P+ variants within lymphoid tissues of mice persistently infected from birth. To map the structural basis of the phenotype, the complete RNA sequence of LCMV ARM 53b (CTL+) was compared with that of its variant ARM clone 13 (CTL-). Differences in 5 of 10,600 nucleotides were found. Three changes are noted in the large L RNA segment, and two are noted in the small S RNA segment. Only two of the changes distinguishing CTL+ from CTL- isolates affect amino acid coding: lysine to glutamine at amino acid 1079 of the polymerase protein, and phenylalanine to leucine at amino acid 260 of the envelope glycoprotein (GP). We also analyzed two additional CTL- variants and four spontaneous CTL+ revertants. All three CTL- variants differ from the original CTL+ parental strain at GP amino acid 260, indicating that this amino acid change is consistently associated with the CTL- phenotype. By contrast the other four mutations in LCMV are not associated with the CTL- phenotype. Sequence analysis of the coding regions of four CTL+ revertants of ARM clone 13 did not reveal back mutations at the GP 260 locus. This finding indicates that the GP 260 mutation is necessary but not sufficient for a CTL- P+ phenotype and that the reversion to CTL+ P- is likely either due to secondary mutations in other regions of the viral genome or to quasispecies within the revertant population that make significant contributions to the phenotype.     

Susceptibility of inbred Syrian golden hamsters (Mesocricetus auratus) to lethal disease by lymphocytic choriomeningitis virus

An acutely lethal LCMV disease model has been established in the Syrian golden hamster (Mesocricetus auratus) in which lethality and disease are dependent upon both the inbred hamster strain and the LCMV strain. Young adult inbred, male and female, hamsters were tested for lethal-disease susceptibility by lymphocytic choriomeningitis virus (LCMV) strains, WE or Armstrong (ARM). With WE inocula, PD4 and MHA inbred hamsters were highly susceptible to a wasting disease. LVG and LHC inbred hamsters were intermediate in susceptibility; some of these animals died of wasting illness, and others exhibited minimal disease and survived. CB and LSH hamsters were highly resistant to any disease by WE. Mean survival times of susceptible hamsters given lethal WE inocula approximated 2.5 weeks and were not dependent on virus dose. By 1.5 weeks after WE inoculation wasting disease signs were notable and consisted of lethargy, progressive body weight loss, and diarrhea. The LCMV strain, ARM, was avirulent for all hamster strains, causing neither death nor disease. Hamsters surviving WE or ARM inoculation appeared healthy, produced LCMV antibody, and acquired resistance to further lethal WE challenge. Despite hamster-lethality differences. WE and ARM appeared comparably immunogenic for all hamster strains, based on host antibody titers. A number of other differences between the LCMV strains were, however, noted which could be relevant to virus virulence and lethality for hamster hosts. These included guinea pig lethality, temperature sensitivity, and plaque morphology.     

Polymorphism of cytotoxic T-lymphocyte clones that recognize a defined nine-amino-acid immunodominant domain of lymphocytic choriomeningitis virus glycoprotein

To assess the heterogeneity of cytotoxic T lymphocytes (CTLs) directed against viral epitopes, we studied six class I major histocompatibility complex-restricted (H-2Db) CTL clones that recognize the same 9-amino-acid immunodominant epitope, amino acids 278 to 286 from envelope glycoprotein 2 (GP2) of lymphocytic choriomeningitis virus (LCMV). Using Southern blot analysis of beta-chain rearrangements, we found that each clone has a unique restriction pattern, providing evidence of the independent derivation of the clones and suggesting that the clones express different beta-chain sequences for their T-cell receptor. All these clones killed syngeneic target cells infected with strain Armstrong or WE of LCMV; however, two of the six clones failed to recognize target cells infected with the Pasteur strain of LCMV. Sequence analysis of LCMV Armstrong, WE, and Pasteur GP in the region of amino acids 272 to 293 demonstrated a single-amino-acid substitution at amino acid 278 in the region of the defined epitope in the Pasteur strain. Interestingly, one of the two CTL clones that failed to lyse LCMV Pasteur-infected target cells nevertheless efficiently and specifically killed uninfected target cells coated with the appropriate LCMV Pasteur peptide, while the other clone failed to do so. This indicated a dichotomy between processing of the synthesized protein initiated by infection and a peptide exogenously applied. Dose-response studies utilizing several peptides with substitutions in GP amino acid 278 indicate that CTL recognition occurs at the level of a single amino acid and suggest that this difference is likely recognized at the level of the T-cell receptor.     

Genomic analysis and pathogenic characteristics of lymphocytic choriomeningitis virus strains isolated in Japan

Lymphocytic choriomeningitis virus (LCMV) is a zoonotic pathogen of which mice are the natural reservoir. Different strains and clones of LCMV show different pathogenicity in mice. Here we determined the complete genomic sequences of 3 LCMV strains (OQ28 and BRC which were isolated from mice in Japan and WE(ngs) which was derived from strain WE). Strains OQ28 and BRC showed high sequence homology with other LCMV strains. Although phylogenetic analyses placed these 2 Japanese strains in different subclusters, they belonged to same cluster of LCMV isolates. WE(ngs) and WE had many sequence substitutions between them but fell into same subcluster. The pathogenicity of the 3 new LCMV isolates was examined by inoculating ICR mice with 10² and 10⁴ TCID₅₀ of virus. ICR mice infected with OQ28 or WE(ngs) exhibited severe clinical signs, and some of the infected mice died. In contrast, all ICR mice infected with BRC showed no clinical signs and survived infection. Virus was detected in the blood, organs, or both of most of the surviving ICR mice inoculated with either OQ28 or WE(ngs). However, virus was below the level of detection in all ICR mice surviving infection with strain BRC. Therefore, LCMV strains OQ28 and BRC were genetically classified in the same cluster of LCMV strains but exhibited very different pathogenicity.Abbreviations: dpi, days postinfection; GP, viral glycoprotein; h, hydrophobic region; IFA, indirect fluorescent antibody assay; L, viral RNA-dependent RNA polymerase; LCMV, lymphocytic choriomeningitis virus; NP, nucleocapsid protein; UTR, untranslated region; Z, zinc-finger proteinLymphocytic choriomeningitis virus (LCMV) is a member of the genus Arenavirus in the family Arenaviridae. The genus Arenavirus is divided into 2 groups (Old World and New World arenaviruses) according to genetic and antigenic characteristics.⁴ LCMV is a member of the Old World arenavirus group, which also includes Lassa, Mopeia, Mobala, and Ippy viruses.^4,10 The LCMV genome contains 2 negative-sense single-stranded RNA segments, designated S RNA and L RNA, with approximate sizes of 3.4 kb and 7.2 kb, respectively.^30,31 Each RNA segment has an ambisense coding strategy, encoding 2 different proteins in opposite orientations. S RNA encodes the nucleocapsid protein and glycoprotein, and L RNA encodes the viral RNA-dependent RNA polymerase and a small zinc finger protein.^25,30LCMV is a zoonotic agent that is transmitted to humans via urine or saliva of infected mice (Mus musculus), which are a natural reservoir of the virus.⁴ The prevalence of LCMV in mice is 7.0% to 25.9% in Japan and 4% to 9% in Europe.^{5,17,19,20,35} Mice are naturally infected by either vertical or horizontal transmission of the virus, and infected mice usually show no clinical signs. In contrast, experimentally infected mice inoculated intraperitoneally or intracerebrally can exhibit clinical signs such as ruffled fur, half-closed eyes, hunched posture, immobility, and neurologic deficits.^4,12,19 Although human LCMV infections are generally either asymptomatic or mild, immunodeficient persons can develop spontaneous abortion, severe birth defects, aseptic meningitis, or fatal infections.^1,2,13,22,27 Therefore, LCMV is an important agent that should be monitored in facilities housing and breeding mice.LCMV strains Armstrong, Traub, and WE were isolated during the 1930s from laboratory mice and humans working in a mouse facility.⁴ Many other LCMV strains and clones used in research originated from these 3 isolates. Strains Aggressive and Docile are clones (variants) of strain UBC, which was derived from the parental strain WE, and strains E350, CA1371, 53b, and clone 13 were all derived from strain Armstrong.⁴ The lethality of strains Aggressive and Docile varies between mouse strains.³⁸ Mice inoculated with 53b develop acute infections, whereas those inoculated with clone 13 mount chronic infections, even though both of the strains were derived from strain Armstrong.²⁹ Furthermore, strain Armstrong produces more severe disease in C3H mice than do strains WE and Traub.⁴ Therefore, previous studies indicate that mice infected with different strains of LCMV exhibit differences in clinical signs and lethality.^4,7 LCMV is a noncytolytic virus and causes immune-mediated viral disease.¹² The clinical signs and lethal disease arise because virus-specific T cells attack infected cells on critical organs in infected mice.¹²Here we report the characterization of 2 LCMV strains recently isolated in Japan (strains OQ28 and BRC) and a passaged isolate of strain WE. The complete genomic sequences of these 3 strains were determined, and their phylogenetic relationship to other LCMV strains was assessed. We also evaluated the pathogenicity in ICR mice of these isolates.     

Genetic analysis of in vivo-selected viral variants causing chronic infection: importance of mutation in the L RNA segment of lymphocytic choriomeningitis virus.

Viral variants with different biological properties are present in the central nervous systems (CNS) and lymphoid tissues of mice persistently infected with lymphocytic choriomeningitis virus (LCMV). Viral isolates from the CNS are similar to the original Armstrong LCMV strain and induce potent virus-specific T-cell responses in adult mice, and the infection is rapidly cleared. In contrast, LCMV isolates derived from spleens of carrier mice cause persistent infections in adult mice. This chronic infection is associated with low levels of antiviral T-cell responses. In this study, we genetically characterized two independently derived spleen variants by making recombinants (reassortants) between the spleen isolates and wild-type (wt) LCMV and showed that the ability to persist in adult mice and the associated suppression of T-cell responses segregates with the large (L) RNA segment. In addition, we analyzed a revertant (isolated from the CNS) derived from one of the spleen variants. By comparing the biological properties of three reassortants that contained the same S segment but had the L segment of either the original wt Armstrong LCMV, the spleen variant derived from it, or the CNS revertant derived from the spleen variant, we were able to show unequivocally that biologically relevant mutations occurred in the L segment not only during generation of the spleen variant from wt LCMV but also in reversion of the spleen variant to the wt phenotype. Thus, our results showed that (i) genetic alterations in the L genomic segment were involved in organ-specific selection of viral variants, and (ii) these mutations profoundly affected the ability of LCMV to cause chronic infections in adult mice.     

cDNA sequence analysis confirms that the etiologic agent of callitrichid hepatitis is lymphocytic choriomeningitis virus.

Callitrichid hepatitis is an infection of New World primates caused by an arenavirus, currently referred to as callitrichid hepatitis virus, that is closely related to lymphocytic choriomeningitis virus (LCMV). We have cloned and sequenced the GP-C gene of callitrichid hepatitis virus and found that the cDNA sequence is 84 to 86% identical to those of the GP-C genes of LCMV strains Armstrong and WE, while the deduced amino acid sequence is 95 to 96% identical to those of the GP-C gene products of the same strains. This high degree of similarity indicates that the etiologic agent of callitrichid hepatitis is in fact LCMV. The wide geographic distribution of callitrichid hepatitis outbreaks in the United States serves as a reminder that LCMV is also a human pathogen whose public health implications are not well understood.     

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus. I. Generation and recognition of virus strains and H-2b mutants

Cytotoxic T lymphocytes (CTL) were induced in C57BL/6 and (C57BL/6 X DBA/2)F1 mice after immunization with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV-Arm) and were cloned by limiting dilution in vitro. The cytotoxic activity of these clones was LCMV specific and H-2 restricted. All clones induced in C57BL/6 (H-2b) mice with LCMV-Arm lysed target cells infected with each of five distinct strains of LCMV (Arm, Traub , WE, Pasteur, and UBC ), suggesting recognition of common regions of viral proteins in association with H-2b molecules. In contrast, one clone obtained from (B6 X D2)F1 mice and restricted to the H-2d haplotype only lysed cells infected with one of three strains of virus (Arm, Traub , WE) but not two others (Pasteur, UBC ), suggesting recognition of variable regions of viral proteins in the context of H-2d molecules. To assess the fine specificity for H-2 molecules, we tested H-2Kb-restricted CTL clones for their ability to kill LCMV-infected target cells bearing mutations in their H-2Kb, and we tested clones presumed to be restricted to the H-2Db region for their ability to all LCMV targets cells bearing a mutation in the H-2Db region. Several different patterns of killing of the mutant targets were observed, indicating that a number of different epitopes on the H-2b molecules were used as restricting determinants for LCMV antigen recognition by CTL. Thus, cross-reactive viral determinants were recognized in the context of several different restricting determinants. Mutations in the N or C1 domains of the H-2 molecule affected recognition by a single LCMV specific CTL clone. One implication of this result is that CTL recognize a conformational determinant on the H-2 molecule formed by the association of virus antigen(s) with H-2. An alternate explanation is that one site on the H-2 molecule is involved in the interaction of viral antigens with H-2, whereas another may serve as a binding site for the CTL receptor.     

Variants able to cause growth hormone deficiency syndrome are present within the disease-nil WE strain of lymphocytic choriomeningitis virus.

Persistent infection of C3H/St mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong leads to disordered growth and hypoglycemia. Both host and viral determinants contribute to this growth hormone (GH) deficiency syndrome (GHDS). Development of the GHDS correlates with the virus's ability to replicate in the GH-producing cells and cause reduced levels of GH synthesis. LCMV strain WE infects few GH-producing cells and does not cause GHDS in C3H/St mice. We show here that clonal variants isolated from the GHDS-nil WE population are able to replicate at high levels in GH-producing cells and cause GHDS in C3H/St mice. These variants are stably maintained, but phenotypically silent, within the GHDS-nil WE population.     

Altering α-dystroglycan receptor affinity of LCMV pseudotyped lentivirus yields unique cell and tissue tropism

Background

The envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV) can efficiently pseudotype lentiviral vectors. Some strains of LCMV exploit high affinity interactions with α-dystroglycan (α-DG) to bind to cell surfaces and subsequently fuse in low pH endosomes. LCMV strains with low α-DG affinity utilize an unknown receptor and display unique tissue tropisms. We pseudotyped non-primate feline immunodeficiency virus (FIV) vectors using LCMV derived glycoproteins with high or low affinity to α-DG and evaluated their properties in vitro and in vivo.

Methods

We pseudotyped FIV with the LCMV WE54 strain envelope glycoprotein and also engineered a point mutation in the WE54 envelope glycoprotein (L260F) to diminish α-DG affinity and direct binding to alternate receptors. We hypothesized that this change would alter in vivo tissue tropism and enhance gene transfer to neonatal animals.

Results

In mice, hepatic α- and β-DG expression was greatest at the late gestational and neonatal time points. When displayed on the surface of the FIV lentivirus the WE54 L260F mutant glycoprotein bound weakly to immobilized α-DG. Additionally, LCMV WE54 pseudotyped FIV vector transduction was neutralized by pre-incubation with soluble α-DG, while the mutant glycoprotein pseudotyped vector was not. In vivo gene transfer in adult mice with either envelope yielded low transduction efficiencies in hepatocytes following intravenous delivery. In marked contrast, neonatal gene transfer with the LCMV envelopes, and notably with the FIV-L260F vector, conferred abundant liver and lower level cardiomyocyte transduction as detected by luciferase assays, bioluminescent imaging, and β-galactosidase staining.

Conclusions

These results suggest that a developmentally regulated receptor for LCMV is expressed abundantly in neonatal mice. LCMV pseudotyped vectors may have applications for neonatal gene transfer.

Abbreviations

Armstrong 53b (Arm53b); baculovirus Autographa californica GP64 (GP64); charge-coupled device (CCD); dystroglycan (DG); feline immunodeficiency virus (FIV); glycoprotein precursor (GP-C); firefly luciferase (Luc); lymphocytic choriomeningitis virus (LCMV); nuclear targeted β-galactosidase (ntLacZ); optical density (OD); PBS/0.1% (w/v) Tween-20 (PBST); relative light units (RLU); Rous sarcoma virus (RSV); transducing units per milliliter (TU/ml); vesicular stomatitis virus (VSV-G); wheat germ agglutinin (WGA); 50% reduction in binding (C₅₀).