Only two related thiamine transporters are known in mammals, THTR1 and THTR2. Here, we have cloned the feline THTR2 cDNA and show that both the feline THTR1 and THTR2 proteins specifically transport thiamine with kinetics and transport rates similar to those of human THTR1. Infection by FeLV-A was associated with disruption of thiamine transport in both feline cells and nonfeline cells engineered to express feTHTR1. Furthermore, feline fibroblast cells infected with FeLV-A did not grow under conditions of limiting thiamine, showing a clear physiologic effect of FeLV-A infection.
The 61E strain of FeLV-A employed in our study only uses THTR1 as a receptor for cell entry, but it is clear that other subgroups of FeLV that evolve in FeLV-A-infected cats can use a wide range of related transporters as receptors (). These include the heme transporters FLVCR1 and FLVCR2 and the inorganic phosphate transporters Pit1 and Pit2. Even these divisions in receptor use between different FeLV subgroups are somewhat arbitrary, as one strain of FeLV has been identified that can utilize THTR1, FLVCR1, or FLVCR2 as a receptor for cell entry (31
). Given this plasticity of receptor use, it would not be surprising if some strains of FeLV can utilize THTR2 as a receptor, leading to further disruption of thiamine uptake.
The expression patterns of feTHTR1 and feTHTR2 that we observed in feline tissues are generally consistent with those of THTR1 and THTR2 orthologs in humans and mice; THTR1 is widely expressed, whereas THTR2 is more restricted in its expression. Ubiquitous expression of feTHTR1 has also been described in cats (40
). We were able to detect feTHTR1 in every cell line and tissue that we tested. The broad tissue distribution of feTHTR1 is consistent with its role in mediating FeLV-A infection of multiple tissues and its broad feline host cell specificity. FeLV-A has been isolated from brain, thymus, lymph node, epithelial cells of the salivary gland, and intestine tissues of infected animals, all tissues in which we detected feTHTR1. Our data indicate that there are cells and tissues in cats that express only one thiamine transport protein, feTHTR1, and we expect that such cells will be more susceptible to the pathogenic effects of FeLV-A infection.
The growth of cells infected with FeLV-A at physiological concentrations of thiamine (median value, ≤30 nM; see Results for calculation) was decreased compared to control cells. Even at concentrations as high as 400 nM, well above what would be expected in a feline host, FeLV-A-infected cells still exhibited decreased growth rates compared to uninfected cells. No difference in growth rate between FeLV-A-infected cells and control cells was seen at very high levels of thiamine (33 μM), indicating that passive diffusion or low-affinity transport of the molecule is sufficient to rescue the defect in thiamine transport at high thiamine concentrations. This may explain why FeLV-A infection does not cause notable effects on cell viability under standard tissue culture conditions, where thiamine levels are high (e.g., 12 μM for DMEM).
The specific pathogenesis of FeLV-A has been difficult to define, as the virus often evolves new receptor specificities associated with new subgroups (5
). This has made it difficult to tease out the specific contribution of FeLV-A to pathogenesis. However, FeLV infection among wild cats of the Felidae
family has provided a window into the pathogenicity of FeLV-A. In a study of the prevalence and importance of known feline pathogens in critically endangered, free-ranging Iberian lynx (Lynx pardinus
), 6 of 11 lynx found to be infected with an FeLV-A subgroup virus (DNA and p27 antigen positive) died in a 6-month period. Death in the study population was strongly correlated with FeLV-A infection (P
< 0.001), no endogenous FeLV sequences were detected in Iberian lynx that might give rise to more pathogenic FeLV recombinants, and no other viral pathogens, including FeLV-B and -C, were detected in the FeLV-infected animals. FeLV infection was associated with anemia, lymphopenia, and neutropenia. Sequencing of the FeLV surface glycoprotein genes in the FeLV-infected animals revealed nearly identical viruses that were closely related to the 61E strain of FeLV-A and most likely arose from virus transmitted from a domestic cat. Similarly, genetic characterization of a Florida panther FeLV outbreak in which five FeLV antigen-positive animals died (42
) revealed the presence of virus related to the domestic cat FeLV-A strain 945, there was no evidence of endogenous FeLV sequences in the panthers, and pathogenic outcomes were related to FeLV infection. In this case, clinical symptoms included lymphadenopathy, anemia, septicemia, and weight loss. These results establish the pathogenic potential of FeLV-A independent of other FeLV strains that arise in domestic cats.
While we found that FeLV-A infection could disrupt thiamine uptake at physiological thiamine concentrations and block cell division, this effect could be reversed in the presence of high levels of extracellular thiamine. Thus, if any of the pathological effects of FeLV-A infection result from such disruption, it may be possible to reverse the pathology by raising the thiamine concentration in extracellular fluids. Indeed, the anemia, pancytopenia, and, to some extent, diabetes observed in humans with defective THTR1 function can be effectively treated, without adverse effects, by oral or parenteral administration of 20 to 100 mg thiamine per day (15
). Likewise, these same diseases, which are observed in THTR1 knockout mice given a low thiamine diet, can be reversed by feeding the mice a standard mouse chow that contains a high level of thiamine (45
). Thus, our results predict that the simple addition of high levels of thiamine to the diet may ameliorate disease in FeLV-infected domestic and wild cats. Furthermore, if immunosuppression seen in FeLV-infected cats is in part the result of disruption of thiamine uptake and resultant poor growth of immune system cells, immune control of FeLV will likely be improved by thiamine supplementation. In combination with vaccination, thiamine supplementation may be particularly useful for preservation of endangered wild cats exposed to FeLV.