SummaryThis study has identified a single amino acid change in the viral glycoprotein that profoundly affects the ability of lymphocytic choriomeningitis virus (LCMV) to persist in its natural host . Adult immunocompetent mice infected with a variant--of the Armstrong strain, spleen isolate clone 13 (svA/svA), harbor virus for several months and exhibit suppressed T cell responses . In contrast, adult mice infected with a reassortant virus (svA/wtA) that contains the L segment of the spleen variant and the S segment of the parental wt Armstrong, make potent LCMV specific CTL responses and clear the infection within 2-4 wk . These two viruses, spleen variant clone 13 and the reassortant svA/wtA, are identical in their noncoding regions and show no amino acid changes in any of their viral genes except for one substitution in the glycoprotein . The reassortant virus svA/wtA has a phenylalanine at amino acid residue 260 of the glycoprotein, whereas the spleen variant clone 13 has a leucine at this position . This study constitutes one of the first reports defining the genetic basis of viral persistence at the whole animal level, and identifying a single mutation that markedly increases the ability of a virus to persist in its natural host . S uccessful resolution of a viral infection depends upon a critical balance between the extent of viral spread and replication, and the magnitude of the host's immune response. We have been studying infection of mice with lymphocytic choriomeningitis virus (LCMV)t as a model system to understand the host and viral determinants that lead to viral clearance or persistence (1-4) . Infection of immunocompetent adult mice with the Armstrong strain of LCMV induces a potent antiviral T cellresponse and virus is eliminated within 2 wk . This clearance is mediated by CD8 * virus-specific cytotoxic T lymphocytes (4-7) . In contrast, infection of adult mice with a naturally selected isolate of Armstrong, spleen variant clone 13, results in a disseminated infection, with virus persisting for several months (1, 2). This chronic infection is associated with suppressed T cell responses and susceptibility to opportunistic infection (1, 2, 8) .The LCMV genome consists of two segments of singlestranded RNA, a large (L) segment of 7 .2 kb and a small (S) segment of 3 .4 kb (9-12) . The L RNA segment codes for a large protein, L (molecular mass 250 kD), that is be-1 Abbreviations used in this paper. L, large; LCMV, lymphocytic choriomeningitis virus; S, small; sv, spleen variants; wt, wild type.lieved to be the viral polymerase, and also contains a second open reading frame, designated Z, that encodes for a protein of "10-12 kD. The S segment codes for the three major structural proteins : the internal nucleocapsid protein (NP ; 63 kD) and the two surface glycoproteins GP-1 (43 kD) and GP-2 (36 kD) that are derived from a common precursor polypeptide, GP-C. After coinfwtion of cells with two different LCMV strains, recombinants are generated by reassortment of genome segments. This permits genetic a...

This study documents that the immunosuppressive lymphocytic choriomeningitis virus (LCMV) variant, clone 13, shows a specific predilection for enhanced infection of macrophages both in vitro and in vivo and that single amino acid changes in the viral polymerase and glycoprotein are responsible for macrophage tropism. The growth difference seen between variant clone 13 and the parental Armstrong strain was specific for macrophages, since both clone 13 and Armstrong grew equally well in fibroblasts and neither isolate infected lymphocytes efficiently. Complete sequencing of the clone 13 genome, along with genetic analysis, showed that a single amino acid change in the polymerase (K->Q at position 1079) was the major determinant of virus yield in macrophages. This was proven unequivocally by comparing the sequences of parental and reassortant viruses, which were identical at all loci except for the single mutation in the polymerase gene. This finding was further strengthened by showing that reversion at this site back to lysine (Q-K) resulted in loss of macrophage tropism. In addition, an independently derived macrophage-tropic variant of LCMV, clone 28b, had a K->N mutation at the same position. Thus, these results show that substitution of the positively charged amino acid K with a neutral amino acid (either Q or N) at residue 1079 of the polymerase resulted in enhanced viral replication in macrophages. In addition to the polymerase change, a mutation in the glycoprotein was also associated with macrophage tropism. This single amino acid change in the glycoprotein (F->L at position 260) did not affect virus yield per macrophage but was critical in determining the number of macrophages infected. Our previous studies have shown that the same two mutations in the polymerase and glycoprotein are essential for establishing a chronic infection in adult mice. Since the same mutations confer macrophage tropism and ability to persist in vivo, these studies provide compelling evidence that infection of macrophages is a critical determinant of viral persistence and immune suppression.

This study demonstrates cell-specific selection of viral variants during persistent lymphocytic choriomeningitis virus infection in its natural host. We have analyzed viral isolates obtained from CD4+ T cells and macrophages of congenitally infected carrier mice and found that three types of variants are present in individual carrier mice: (i) macrophage-tropic, (ii) lymphotropic, and (iii) amphotropic. The majority of the isolates were amphotropic and exhibited enhanced growth in both lymphocytes and macrophages. However, some of the lymphocyte-derived isolates grew well in lymphocytes but poorly in macrophages, and a macrophage-derived isolate replicated well in macrophages but not in lymphocytes. In striking contrast, the original wild-type (wt) Armstrong strain of lymphocytic choriomeningitis virus that was used to initiate the chronic infection and from which the variants are derived grew poorly in both lymphocytes and macrophages. These three types of variants also differed from the parental virus in their ability to establish a chronic infection in immunocompetent hosts. Adult mice infected with the wt Armstrong strain cleared the infection within 2 weeks, whereas adult mice infected with the variants harbored virus for several months. These results suggest that the ability of the variants to persist in adult mice is due to enhanced replication in macrophages and/or lymphocytes. This conclusion is further strengthened by the finding that the variants and the parental wt virus grew equally well in mouse fibroblasts and that the observed growth differences were specific for cells of the immune system.

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.

Lymphocytic choriomeningitis virus clone 13 (LCMV clone 13), a variant isolated from the spleens of neonatally infected mice, causes persistent infections in mice infected as adults. Such persistently infected mice succumb to a normally sublethal dose of Histoplasma capsulatum, and their macrophages contain overwhelming numbers of yeast cells of the fungus. Both LCMV clone 13 and H. capsulatum yeast cells target and replicate in macrophages of the host. We sought to study the effects of LCMV clone 13 on the ability of macrophages to control growth of H. capsulatum in vitro. We show that the growth of H. capsulatum within macrophages was not directly affected by the presence of LCMV clone 13. However, macrophages containing LCMV clone 13 did not respond fully to gamma interferon (IFN-␥) stimulation. Such unresponsiveness resulted in proliferation of the fungus within macrophages cultured in the presence of IFN-␥. The addition of anti-IFN-␣/␤ antibodies to LCMV clone 13-infected macrophage cultures restored macrophage responsiveness to IFN-␥. These results indicate that production of IFN-␣/␤ by LCMV clone 13-infected macrophages antagonizes their responsiveness to IFN-␥. Such antagonism may be one of the mechanisms by means of which certain viruses cause immune suppression and susceptibility to opportunistic infections.