Lymphotoxins-α (LTα) and -β (LTβ) are members of the tumor necrosis factor (TNF) superfamily and are closely linked to the TNF gene within the major histocompatibility complex of all known mammalian species. LTα polypeptides can form a soluble homotrimer (LT) that is secreted principally by activated T lymphocytes and NK cells, while LTβ polypeptides form type II membrane-bound heterotrimers with LTα polypeptides (LTα₁β₂ or LTα₂β₁) 1. Each form of the trimer is involved in inflammatory responses, induction of apoptosis of target cells, and lymphoid organogenesis.

Hantavirus cardiopulmonary syndrome (HCPS) is caused by several pathogenic New World hantaviruses, but most North American cases of HCPS are caused by Sin Nombre virus (SNV) 2345. The disease is characterized by the production of several proinflammatory cytokines, including LT, in the lungs 6. In contrast, the principal reservoir host of SNV, the deer mouse (Peromyscus maniculatus) 7, exhibits no conspicuous pathology and remains persistently infected for life 89. It is unknown how deer mice can be persistently infected without pathology because few methods have been developed for characterizing cytokine responses in this species. We previously reported a partial cDNA sequence of deer mouse LTα 10 and present here genomic, structural, and phylogenetic characterizations of the complete lymphotoxin genes.

As one of the most ecologically and morphologically diverse groups within Mammalia, Rodentia has always been a challenge to delineate systematically. And, despite the attention the group has received from ecologists and evolutionary biologists, the lineages within the group have been difficult to resolve. In particular, the relationships among members of the family Muridae, arguably the most species-rich family of mammals, have yet to be definitively resolved at the subfamily level and below 11. The introduction and analysis of new sources of molecular data, such as those sampled here, may represent an avenue for understanding the evolution and diversification of the rodents. In addition, the prospect of combining data sets, as examined here, allows investigators a greater suite of options when it comes to understanding the relationships within highly diverse groups such as the Muridae family. Although the taxonomic sampling within this study was limited by the availability of orthologs in GenBank, we have conducted a preliminary examination of the usefulness of LTα and LTβ for phylogeny reconstruction at the subfamily level within the order Rodentia. We present here the extent to which these lymphotoxins (both in separate and combined data sets) have potential for resolving relationships within Muridae.

Lymphotoxin is expressed in fatal cases of HCPS and may contribute to the inflammatory pathogenesis of the disease 6. In lymphocytic choriomeningitis virus infection of New Zealand black house mice, blockade of LTβ receptor reverses respiratory failure and immunopathology 19. It is unknown if lymphotoxin is expressed in the lungs of deer mice infected with SNV. Conspicuous T lymphocyte migration into the lungs of infected deer mice is absent, suggesting that it is not, even though viral RNA can be detected more than 200 days post infection in some deer mice 89. Antigen-specific T cells from the spleens of persistently-infected deer mice appear to be regulatory T cells 20, suggesting the lymphotoxin pathway is not used during persistence.

The TNF family is a frequent target of viral evasion strategies 21 and given the role of these proteins in inflammation they may also be targets of hantaviruses in their rodent hosts, which do not exhibit inflammatory responses. LT has been implicated in host-virus coexistence with human and mouse cytomegaloviruses by inducing IFNβ expression in fibroblasts without apparent cytotoxicity or induction of apoptosis 22. In this manner, the expression of LT permits virus persistence without cytopathology. Since hantaviruses and rodent hosts have coevolved for tens of millions of years, it is likely that each has adapted to induce minimal pathology. Since deer mouse CD4⁺ T cells are not susceptible to SNV infection 20 it is unlikely that viral proteins directly affect the LT pathways of these cells.

Structurally, deer mouse lymphotoxins are highly similar to orthologous polypeptides. Deer mouse LTα is predicted to be composed of 169 residues and LTβ is 302 residues long. Each polypeptide shares 11 secondary domains found in mouse and human lymphotoxins.

The deer mouse LTα and LTβ gene and polypeptide sequences are not conspicuously different from orthologous sequences. We did not obtain flanking information of the Lta gene, despite repeated attempts with primers designed to amplify additional 5' and 3' regions. These regions may be less conserved in deer mice compared to other rodent species, which would make primer design problematic. We were able to obtain some promoter information for the Ltb gene, which shares cis-elements found in the house mouse Ltb gene. One difference is the presence of an Sp1 cis-element in deer mouse. Whether this has functional consequences for Ltb expression will require further investigation since only lymphotoxin-α has been examined in human or rodent hantavirus infections 620. All five of the promoters examined here also possess KLF cis-elements, which are recognized by a number of Sp1-family transcriptional repressors 23. The intron/exon structure of deer mouse Ltb is identical to the other rodents, namely that all have three exons instead of four that is found in human LTB because of a splice donor site in intron 2 of the human gene. It is unusual, though, that while the deer mouse and house mouse have identical sequences at this site, the rat sequence possesses a three-nucleotide deletion. Deer mice (Sigmondontinae) are substantially more divergent than are rats and house mice (both Murinae) 24, suggesting the deletion in the rat gene occurred after the rat/house mouse divergence. Additional 5' and 3' information could provide information about these genes that is limited by our PCR approach. Alternative strategies for obtaining such information would be the use of genomic libraries, such as the recently-produced BAC library for the deer mouse http://bacpac.chori.org/. In addition, the deer mouse genome has recently been selected for sequencing (M. Dewey, pers. comm.), which should facilitate such analyses.

In agreement with many molecular studies on subfamily designations within Muridae 10112526, all of our analyses were able to resolve the separation between the Murinae and the Sigmodontinae. Future tests of LTα and LTβ utility need to add lymphotoxin sequence data from other muroid groups such as Tylonyinae, Neotomyinae, Cricetinae, and Arvicolinae; these subfamilies are particularly critical to sample because they are thought to be closely related to both the murines and the sigmodontines 11. Although the analyses reconstructed Muridae, none of the data sets was able to ally the woodchuck (a member of the rodent family Sciuridae) with the other three rodents; in other words, the analyses were not able to resolve which one of the outgroups (woodchuck or human) was more closely related to the murid rodents. Further sampling within Sciuridae and other groups such as Gliridae and Dipodidae may be necessary to test the utility of these data for resolving family-level relationships within Rodentia. Despite that issue, the moderate level of useful variation that both LTα and LTβ possess, in combination with the high level of bootstrap support that characterized the subfamily-level lineages suggest that further investigation of phylogenetic utility is justifiable. In addition, because the two data sets may be combined with one another, investigators may have yet another approach at hand in resolving relationships that are currently problematic.

Collectively, the tumor necrosis factor cluster is nearly identical between mammalian species (Fig. 6; 27). While intervening sequences between Tnf, Lta and Ltb are not complete for deer mice, the structures of these genes are homologous between species. While unlikely, it is possible that gene orientation, order and location of the deer mouse genes are not syntenic to other species' TNF loci. The locus is found in the MHC in all mammalian species that have been examined; however, the chromosomal location of the deer mouse MHC has not yet been determined, but may reside on chromosome 21 (R. O'Neill, pers. comm.).

These sequences represent the fourth rodent, and first New World murid, Lta and Ltb genes described thus far. They exhibit substantial sequence conservation to orthologous sequences and the availability of these sequences may provide insight into their functions in deer mice infected with SNV.

What potential do these molecules hold for phylogeny reconstruction and/or hypothesis testing? The results suggest that these molecules inform on the intrafamilial level within Rodentia; in addition, our findings indicate that combining the LTα and LTβ data into a single data set is appropriate. Thus, LTα and LTβ may hold promise for resolving subfamily relationships not only within Rodentia but within other mammalian orders as well. The implementation of broader taxonomic representation will allow for a fuller test of the utility of these molecules for phylogenetics.

Liver DNA was extracted (PureGene DNA Isolation Kit, Gentra Systems, Minneapolis, MN, USA) from an adult deer mouse (P. maniculatus sonoriensis) 28 and used to amplify fragments representing the Lta and Ltb genes by PCR (PCR Core Kit, Qiagen, Valencia, CA, USA) with primers from highly conserved regions of mouse, rat and human orthologs. The fragments were cloned into pGEM-T Easy vector (Promega, Madison, WI, USA) and transformed into Novablue cells (Novagen, Madison, WI, USA). Plasmids containing inserts of the appropriate sizes were used for DNA sequencing. After initial sequencing runs using T7 and SP6 primers, insert-specific primers were used for primer-walking sequencing. The sequence files were assembled with Sequencher version 4 (GeneCodes, Ann Arbor, MI, USA) and submitted to blast to identify genes 29.

The Lta gene was amplified and cloned using a single primer set (forward, 5'-TGT CTT CCG CTG TGT GCC CC-3'; reverse, 5'-GTG AGA GCT CTG GGT CTG TTT GG-3'). The Ltb gene was cloned from 5' and 3' overlapping PCR products using primers designed from conserved orthologous sequences (5' end, forward 5'-TTC CCC AGT TCA GAG AAC C-3'; reverse 5'-TCC CTC TCC TGT AGT CCA CC-3': 3' end, forward 5'-GAG GCG AGC CAA GAA GAA GC-3'; reverse 5'-ACG ATT CAC ACA TTC GCA CC-3'). Promoter analysis was conducted using NIH Signal Scan database http://www-bimas.cit.nih.gov/molbio/signal/ (Prestridge, D.S. 1991).

LT: lymphotoxin; LTα: lymphotoxin-α subunit; LTβ: lymphotoxin-β subunit.

TR cloned and sequenced the Ltb gene; JP cloned and sequenced the Lta gene. AP conducted the phylogenetic analyses; TS provided the conceptual framework for the project and wrote the manuscript.