These studies focused on the therapeutic target Site 1 Protease by utilizing the hypomorphic mutant woodrat to evaluate systemic S1P inhibition in vivo. As Mbtps1wrt is the only viable animal model with systemic inhibition of S1P, this reagent provides a powerful means to assess the role of S1P inhibition in vivo. Therapeutically, Mbtps1 is an important potential target in the treatment of hypercholesterolemia and often fatal arenavirus infections. We directly assessed the role for this gene for both the acute and persistent variants of the prototypic arenavirus, LCMV.
Surprisingly, in vivo resistance in this model was observed mainly in chronic infection (Clone 13 infection), without a prominent role in acute infection (Armstrong infection). As expected, this hypomorphic mutation in a gene required for the viral life cycle does result in a decrease in viral titers. However, this decrease is organ and cell type specific. Our interpretation of these data is that persistent viral infection requires infection and viral subversion of specific cell types which are genetically resistant to infection in Mbtps1wrt(i.e. bone marrow cells including monocytes and DCs), implicating an important role for these cell types in persistent versus acute infections. We believe these cells might be more sensitive to the wrt mutation (in contrast to splenocytes or hepatocytes) due to a lower level of S1P activity in cells from the bone marrow. Indeed, we observed lower expression of both Mbtps1 as well as downstream target genes in bone marrow as compared to other compartments. Given the hypomorphic activity of Mbtps1 in wrt, this would further decrease Mbtps1 function below a potentially critical threshold necessary for a productive viral infection in the bone marrow.
In order to confirm the mechanism of protection in wrt
, we applied a genetic test, host complementation. In this test, mutation of the viral proteolytic recognition sequence for Mbtps1
,RRLA to RRRR which can be recognized by the ubiquitous protease Furin, should restore viral fitness in the wrt
host. This restoration of viral replication should be specific to mutations in both the host and viral genes involved. Consistent with this reasoning, we observed specific rescue of Cl13FURIN
viral replication in wrt
bmDC. Moreover, complementation was also observed in vivo.
had increased viral fitness in wrt
as compared to otherwise more susceptible WT mice. Of note, it remains unclear why Cl13FURIN
is ultimately attenuated in vivo.
We have observed many times prior that various engineered viruses which we have produced display severe in vivo
attenuation which was not seen in vitro
(unpublished observations, (Emonet et al., 2009
)). We suspect that the numerous differences in cell type and extracellular milieu in vivo
vs. in vitro
contribute to this phenomenon. Importantly, our observations were specific to the proteolytic cleavage site in the LCMV GPC and the cognate mutation in the protease Mbtps1.
Although the 2 amino acid changes in Cl13FURIN
attenuate the virus in vivo
in an unknown manner, the selective advantage offered by complementing viral proteolytic cleavage with Furin outweighs the cost of utilizing the mutant RRRR site. We confirmed this by directly sequencing viral GPC 5dpi from serum. In all cases we observed no deviation from the engineered protease target sequences in output virus as compared to the sequence in the inoculating virus. Importantly, maintenance of the RRRR recognition site in wrt
serum is despite normal processing in several cell types as well as equivalent productive infection in organs (i.e. spleen and liver) at this time in wrt
as compared to WT mice. Importantly, these findings offer insight towards the importance of cell type specificity when targeting viral processing activity. This is imminently important for HIV/AIDS research regarding protease inhibitors in clinical use.
These studies highlight the importance of studying genes systemically in vivo utilizing a hypomorphic mutation when null mutations are non-viable. In this way, one can compare the relative roles of different cell types in a physiologically relevant situation where a therapeutic target is partially inhibited. When limited to complete nulls many therapeutic targets can no longer be studied secondary to embryonic lethality. In addition, complete target inhibition is physiologically unlikely in real-world settings.
mice in this study we have uncovered two key findings regarding therapeutic targeting of S1P: inhibition of S1P function results is host lymphocyte immunodeficiency and inhibition of S1P is a therapeutically viable option for treatment of persistent arenavirus infection in vivo.
These findings are important given the potential therapeutic role of S1P inhibition for treatment of hypercholesterolemia and often fatal arenavirus infections (Hawkins et al., 2008
; Hay et al., 2007
). Understanding the consequences of systemic inhibition of this therapeutic target is critical in determining its clinical utility.
Regarding our understanding of persistent viral infection, analysis of the wrt mutation has demonstrated two critical findings—the role of cell type specificity in controlling persistent viral infection and the potential therapeutic utility of bmDCs in protection from persistent viral infection.
We have found cell type specific resistance to viral growth in bmDC. In response to this finding as well as growing literature supporting the central role of DC in persistent viral infections and tumor immune escape (Chaput et al., 2008
; Liu et al., 2009
; Oldstone, 2009
; Zwirner et al., 2010
), we tested the therapeutic role of bmDC that are genetically resistant to LCMV challenge. We believe wrt
bmDC are therapeutic in contrast to WT bmDC for two reasons. Firstly, DC express a high level of the arenavirus receptor alpha-dystroglycan (unpublished observations), but after viral entry this becomes a dead end for viral replication in wrt
bmDC, effectively a sink for infectious virus particles. Secondly, we observed basal activation in wrt
bmDC which is not inhibited by Cl13 infection. Being a critical cell type in persistent viral infections this may also contribute to host protection, although as we did not observe augmentation in virus specific peptide stimulation 7dpi, this appears to play a smaller role, if any.
Our results raise an important consideration when transferring DCs therapeutically into patients. There has been much speculation and experimentation in further supplementing a host with DC to combat both infectious and oncologic diseases(Ilett et al., 2010
; Lehman et al., 2010
). Our data highlights that simply boosting the levels of these central immune sentinels may not be sufficient to clear a pathogenic challenge. This is likely secondary to the numerous subversive tactics already in place compromising the host immune response. Rather, optimizing this cell type to resist infectious or oncologic countermeasures, as in our model, may be critical to fully utilize DC and other immune cell types to advance the field of immunotherapy.