Memory is a hallmark of the immune system and ever since its recognition there has been considerable interest in understanding how immunity is maintained. The current model is that long-term memory is dependent on persistent antigenic stimulation. We report here results that challenge this view and provide evidence that antigen is not essential for the maintenance of CD8+ T-cell memory. We show that memory CD8+ cytotoxic T lymphocytes persist indefinitely in the absence of priming antigen, retain the memory phenotype (CD44hi), and provide protection against virus challenge. These findings suggest a re-evaluation of our current thinking on mechanisms involved in maintaining immunity and have implications towards designing effective vaccination strategies.
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.
The molecular cascade of stress response in higher eukaryotes commences in the cytoplasm with the trimerization of the heat shock factor 1 (HSF1), followed by its transport to the nucleus, where it binds to the heat shock element leading to the activation of transcription from the down-stream gene(s). This well-established paradigm has been mostly studied in cultured cells. The developmental and tissue-specific control of the heat shock transcription factors (HSFs) and their interactions with heat shock promoters remain unexplored. We report here that in the rat lens, among the three mammalian HSFs, expression of HSF1 and HSF2 is largely fetal, whereas the expression of HSF4 is predominantly postnatal. Similar pattern of expression of HSF1 and HSF4 is seen in fetal and adult human lenses. This stagespecific inverse relationship between the expression of HSF1/2 and HSF4 suggests tissue-specific management of stress depending on the presence or absence of specific HSF(s). In addition to real-time PCR and immunoblotting, gel mobility shift assays, coupled with specific antibodies and HSE probes, derived from three different heat shock promoters, establish that there is no HSF1 or HSF2 binding activity in the postnatal lens nuclear extracts. Using this unique, developmentally modulated in vivo system, we demonstrate 1) specific patterns of HSF4 binding to heat shock elements derived from ␣B-crystallin, Hsp70, and Hsp82 promoters and 2) that it is HSF4 and not HSF1 or HSF2 that interacts with the canonical heat shock element of the ␣B-crystallin gene.Induced transcription from heat shock promoters is mediated by the activation of transacting HSFs 1 (1, 2). There are four known HSFs (HSF1, HSF2, HSF3, and HSF4). HSF3 is an avian HSF (3, 4). Although yeast and Drosophila melanogaster have a single gene that encodes an HSF, higher eukaryotes, animals, and plants have multiple genes that code for HSFs (4 -6). HSF1 and HSF2 transcription factors have almost identical gene structures (4). The heat shock response starts with the cytoplasmic HSF and its trimerization and transport to the nucleus, where it binds to the heat shock element (HSE) in the heat shock promoter, activating transcription of the down stream heat shock gene(s) (1, 4). Both HSF1 and HSF2 contain three hydrophobic repeats, HR-A, -B, and -C. HR-A and -B are involved in trimerization upon reception of the stress signal. HR-C, located at the carboxyl terminus, has been suggested to inhibit trimerization in the uninduced state. HSF4, on the other hand, does not contain the HR-C domain; it therefore exists as a trimeric unit and binds to the DNA constitutively (for review, see Ref. 4).HSF1 is considered to be the universal HSF and mediates expression of heat shock genes upon reception of a stress signal such as high temperature, whereas HSF2 is associated with developmental control. Although it has not been experimentally established, the assumption in this generalization is that all tissues and cells contain HSF1 as a pre-existing HSF in the cytoplasm to enab...
Viral variants of different phenotypes are present in the central nervous system (CNS) and lymphoid tissues of carrier mice infected at birth with the Armstrong strain of lymphocytic choriomeningitis virus. The CNS isolates are similar to the parental virus and cause acute infections in adult mice, whereas the lymphoid isolates cause chronic infections associated with suppressed T-cell responses. In this study, we provide a molecular basis for this organ-specific selection and identify a single amino acid change in the viral glycoprotein that correlates with the tissue specific selection and the persistent and immunosuppressive phenotype of the variants. This phenylalanine (F)-to-leucine (L) change at position 260 of the viral glycoprotein was seen in the vast majority (43 of 47) of the lymphoid isolates, and variants with L at this residue were selected in spleens of persistently infected mice. In striking contrast, isolates with the parental sequence (F at residue 260) predominated (48 of 59 isolates) in the CNS of the same carrier mice. Complete nucleotide sequence analysis of the major structural genes of several independently derived (from different mice) spleen isolates showed that these variants were >99.8% identical to the parental virus. In fact, the only common change among these spleen isolates was the F-*L mutation at residue 260 of the glycoprotein. These results show that an RNA virus can exhibit minimal genetic drift during chronic infection in its natural host, and yet a single or few mutations can result in the organ-specific selection of variants that are markedly different from the parental virus.
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