At least three CD209
family members have appeared during primate evolution, including the novel CD209L2
gene identified in OWM and nonhuman apes. Tandem arrangement of CD209
family members has been observed consistently across species, as human CD209
and the five mouse CD209
homologues are located adjacent to one another on human chromosome 19 and mouse chromosome 8, respectively (2
). Further, we have established that rhesus monkey CD209
map to a single BAC clone, indicating their close physical proximity (data not shown). Given the relative positions of CD209
in humans, as well as CD209
in rhesus monkeys, it is likely that all three genes present in nonhuman apes are also adjacent to one another.
The combination of CD209 and CD209L2 in OWM and apes except for humans suggests that these two genes were present in a common primate ancestor at least 20 million years ago and that through some genetic mechanism, such as unequal crossing over, CD209L2 was lost in the human lineage. CD209L1, which is missing in OWM, may have arisen by gene duplication in a common ancestor of the apes approximately 20 million years ago, but if so, comparisons of the gene sequences do not conclusively indicate whether CD209L1 was most likely derived by duplication of CD209 or CD209L2. On the other hand, CD209L1 may be more ancient than CD209 or CD209L2 if mutation rates are assumed to be constant for all three genes, since phylogenetic analysis suggests that CD209L1 is more distantly related to CD209 and CD209L2 than CD209 and CD209L2 are to each other. In this case, an ancestor of OWM would have to have lost the CD209L1 gene. Sequence data from the CD209 gene family members in prosimians and NWM, which appear to be somewhat divergent on the basis of our inability to amplify these genes with the primers described herein, may provide further insight into the evolutionary history of this gene family in primates.
The 69-bp repeats encoding the neck region of the CD209 family molecules have also been subject to genetic mechanisms resulting in insertions or deletions. The wide range of repeats observed for CD209L1 in both humans and chimpanzees implies that if its gene product is essential, then the physiologic function of CD209L1 is not very sensitive to the length of its neck region. The absence of CD209L1 in OWM, as well as its possible pseudogene status in orangutans, questions the necessity of this gene in the ape species that actually express the gene. If, indeed, this molecule is highly beneficial in species that express it, then such functions may have developed in recent evolutionary history. Alternatively, CD209L1 and CD209L2 may share functional characteristics and the absence of one may be compensated for by the presence of the other. The conserved nature of the region encoding the CD209L2 neck is most reasonably explained by the occurrence of a severe deletion that became fixed. Because of its nonrepetitive nature (it contains only a fraction of the 69-bp repeat), it may subsequently have become resistant to processes resulting in insertions or deletions. Like CD209L1, the functional necessity of CD209L2 is uncertain given its absence in humans and the possibility that it is a pseudogene in gorillas. Finally, the conserved nature of the human CD209 repeat region is curious in light of its somewhat polymorphic nature in other ape species. If, indeed, this region of the CD209 gene family is prone to expansion and contraction in general, then the specific neck length of human CD209 must confer a particularly favorable function, or unique structural characteristics of the DNA in the region of the human CD209 gene have evolved that inadvertently inhibit repeat insertions or deletions. The presence of this gene in OWM and apes, as well as the ability of its protein product to bind ICAM-3 at relatively high levels, may suggest that it has maintained a function in cell-cell interaction over millions of years.
Several studies suggest the importance of neck region length for efficient virus transmission to target T cells. The mouse CD209 molecule, referred to as DC-SIGN (1
) or SIGNR1 (17
), which contains a short neck region of roughly 2.5 repeat lengths, has been shown to bind HIV-1, but it is inefficient in virus transmission to T cells (1
). Transfection studies with a human CD209
construct that lacks the entire coding sequence for the neck region indicated that this segment is essential for both binding of HIV-1 and efficient virus transmission by CD209 (18
). Thus, the inefficient virus transmission observed when cells expressing Mm-CD209L2 were used could be explained in part by its truncated neck domain, although other parts, such as the cytoplasmic tail, may also influence the process. Whether the poor virus transmission is caused by a weak interaction between the viral envelope and CD209L2 remains to be determined. However, virus binding and transmission by CD209 are dissociable functions (19
), and it is possible that the structural differences between CD209 and CD209L2 specifically affect the process of virus transmission by CD209L2 while maintaining strong binding of the virus. Further investigation of the mechanistic basis of the poor transmission of HIV-1 or SIV by CD209L2 should involve detailed analysis of both virus binding and transmission by using various chimeric and mutant constructs. Given that CD209 and CD209L1 can form tetramers via their neck domains (16
) and Mm-CD209 and Mm-CD209L2 may be coexpressed, it will also be interesting to examine whether CD209L2 expression has dominant negative or regulatory effects on CD209-mediated virus transmission.
Patterns of Mm-CD209
expression are similar to those of human CD209L1
in that it is expressed most notably in the liver and LN (Fig. ), the only tissues containing mRNA for CD209
homologues detectable by Northern analysis. The more sensitive RT-PCR method indicated that a number of additional tissues also express the molecule but evidently at lower levels. Insufficient amounts of mRNA from skin prohibited its use in Northern analysis, but strong signals representing CD209
were observed by RT-PCR in spite of nearly undetectable levels of the G3PDH
control (Fig. ). Thus, skin may also express significant amounts of the CD209
homologues. Previous studies have indicated the expression of Mm-CD209 by DCs and macrophages in lymphoid and mucosal tissues, as well as by sinusoidal endothelial cells in the liver (10
); however, given the possibility that antibodies raised against Mm-CD209 cross-react with CD209L2, it is necessary to determine which molecule(s) is responsible for the observed staining patterns.
Expression of the rhesus genes in inguinal LN was detectable by RT-PCR but not by Northern analysis, whereas their expression in Ax-LN was relatively pronounced and readily detectable by Northern analysis. This observation may reflect unique properties specific to secondary lymphoid tissues differentially located in the body. For example, during an early response to SIV infection, rhesus LN in the upper body, such as Ax-LN, are activated relative to those located in the lower body, such as inguinal LN (26
The level of Mm-CD209
expression may vary among individuals (Fig. ), although a more thorough analysis involving larger sample sizes is required in order to define the factors that may influence this heterogeneity (e.g., age, disease status, polymorphisms, etc.). Rhesus MDDCs have been shown to lack expression of CD209 (27
), and we now infer that these cells also failed to express CD209L2 since the probes and MAbs used in the previous analyses recognize both family members. Given the possibility of heterogeneity in CD209
family gene expression among different individuals or under different conditions within a single individual, reassessment of MDDCs may be warranted.
Insight regarding the differential biological functions of the CD209 family of molecules may explain the fluctuation in the number and type of CD209 family genes observed across primate species. If CD209 is truly important in enhancing HIV pathogenesis by facilitating infection of T cells in vivo, selection for deleterious mutations in CD209 may occur (particularly in regions of the world where HIV infection is highly endemic), potentially leading to the demise of what is currently the most stable CD209 gene family member.