Restrictions on the ability of virus to migrate freely between different cells, tissues, and organs can have a profound effect on viral diversity and divergence, the ability of virus to replicate (or persist in a dormant form) during antiretroviral therapy, and the ability of virus to acquire new cell tropisms. Previous studies 
have suggested that the lung is a privileged site in which HIV-1 evolves quickly towards a homogeneous phenotype. In this study, we have sought to test these predictions by sequencing virus from a large number of paired samples from blood and lung. While we did find evidence for compartmentalization between lung and at least one blood tissue in ten of 18 subjects, compartmentalization was actually quite limited in this data set. In general, HIV-1 variants from the blood and lung were more often intermixed than segregated; in most of the subjects evidence of compartmentalization was not apparent from visual inspection of the phylogenetic trees. Furthermore, we saw no evidence that lung samples contained signature sites in the V3 region or an excess of CCR5-tropic viruses or archival genotypes. The limited evidence of compartmentalization was observed in HIV-1 patients with (Indiana cohort) or without (Seattle cohort) evidence of elevated lung lymphocyte counts, and was not notably influenced by whether virus came from induced sputum, BAL fluid, BAL cells, or BAL cell samples enriched for AM.
The limited amount of compartmentalization that we did observe seemed to be related to two factors. The first was the unexpected presence of identical or nearly identical sequences in some of the BAL samples. Although identical sequences have been observed in samples from patients on HAART 
, it is rare to find identical env
sequences from blood samples from patients not on therapy. In the case of UW-BAL-06, 20 of the 22 BAL sequences were identical or nearly identical, despite avoiding resampling and contamination. Many other subjects had from two to six identical or nearly identical lung sequences. The presence of nearly identical sequences contributed substantially to statistical evidence of compartmentalization in these subjects, and suggests that compartmentalization could be due, in part, to small, localized clonal expansions as opposed to restrictions in migration 
The second factor underlying compartmentalization in our study was a correlation between compartmentalization and plasma RNA. Detecting differences between any proviral tissue and plasma is not surprising, as virus found in plasma was sequenced from HIV-1 RNA, while the virus found in lung tissue, PBL, PBM, and PBMC were sequenced from viral DNA. It has been shown that virus in plasma may reflect a more contemporary quasispecies, in which the cellular origins of the plasma virus are most likely from an actively replicating cell population which turns over very quickly (perhaps from a small subset of the PBMC pool), while cell-associated DNA may harbor archival species derived from latently infected, long-lived cells within the PBMC pool 
. These differences in turnover rate could be substantial enough to look like compartmentalization, and could therefore be contributing to the amount of blood/lung compartmentalization detected.
By contrast, we did not find evidence that compartmentalization was related to selection for specific viral variants. If antibody escape mutants were driving compartmentalization between lung and blood we might expect to see differing PNGS and length profiles, as HIV-1 variants in the lung would have access to differing cellular compositions and antibody profiles, and would adapt accordingly. Our finding of both X4 and R5 viruses in lung are consistent with previous studies 
, which found both SI and NSI genotypes in a variety of tissues, including lung (though they also reported tissue-specific viral variants in brain, lung, and testis).
Lung tissue is distinguished from other tissues in having a very high proportion of macrophages, and it has been proposed that selection for infection of macrophages might create distinguishing genetic features of lung virus compared to blood virus 
. Studies have shown that AM, which are the frontline of defense against pathogens in the lung, can be productively infected by HIV-1 
, though potentially at low levels 
. The presence of AM as local targets for HIV-1 infection, along with an environment containing a variety of antimicrobial proteins and peptides that comprise the pulmonary innate immune response 
, could theoretically drive selection for specialized HIV-1 quasispecies within the lung. The fact that HIV-infected AM can survive and produce virus for several weeks in vitro
further suggests that the lung could contain an excess of archival genotypes (viruses that genetically resemble viruses found in lymphocytes during earlier phases of infection).
However, the above theoretical reasons for expecting that the lung could be a reservoir for distinct viral populations need to be balanced against findings that the frequency of detecting HIV-1 in alveolar lymphocytes is much higher than the frequency of detecting HIV-1 in AM in individuals not undergoing ART 
, which could indicate trafficking of lymphocytes from the peripheral blood to the lung 
. Furthermore, CD4+ T cells in BAL are infected at similar frequencies to CD4+ T cells found in blood in subjects with chronic infection, and are not massively depleted, as has been found in the GI tract 
. Also, it is known that both cell-free virions and HIV-infected lymphocytes are capable of trafficking between blood and lung 
, and detection of HIV-1 in AM is more likely in patients with more advanced disease and lower peripheral blood CD4+ T cell counts 
HIV-1 variants in lung have previously been found to be similar to virus found in lymphoid tissue in some subjects 
. The lymphatic and circulatory systems continually circulate cells throughout the body, and could be vehicles for free virus and/or HIV-infected T-cells trafficked between lymph nodes and the lung. These processes may be accelerated as T lymphocytes are trafficked into the lung in response to inflammatory reactions, and indeed, HIV-1 infection induces T-cell alveolitis in the lung during early infection 
. While the relatively small subset of CD4+ T cells found in the lung are infected in high frequencies, it has been reported that CD8+ T cells, present in high quantities during HIV-induced alveolitis, can also be infected 
. High densities of CD4+ T-cells or CD8+ T-cells may create microenvironments for rapid spread of viruses between lymphocytes and alveolar macrophages. Whatever the mechanism, our results support a picture of continuous communication between circulating blood and lung tissue, with a limited degree of localized evolution or clonal replication.
We cannot exclude the possibility that the lung could serve as a reservoir for HIV-1 in some cases. For instance, suppression of viral load (such as in subjects on effective therapy) may allow for minority cell populations within the lung to sustain populations of viruses that are distinct from those found in blood. Indeed, it has been shown in pediatric subjects under mostly effective ART (<50 copies/mL with blips <400 copies/mL) that virus in induced sputum evolves more drug resistance mutations than virus obtained from blood, and also that viral env
gene sequences in induced sputum is more diverged than virus in blood 
. This suggests that viral suppression may enable detection of low-level replication in the lung in the absence of widespread dissemination of virus from blood, and that effective suppression of virus could allow for restriction of migration and be a complicating factor in treatment. However, we did not see the same pattern in our study; two IS subjects had viral load <50 (IS-8247 and IS-8886) and one IS subject had viral load of 52 copies/mL (IS-9000) due to effective ART (), and divergence did not differ significantly between lung and blood in any of these subjects (). This suggests the increased divergence in lung described here could be a phenomenon associated with early successful treatment and perhaps limited to pediatric subjects, though we acknowledge we did not sample enough subjects on effective ART to investigate this issue thoroughly.
In summary, the extensive intermingling of virus from multiple blood tissues with virus from the lung, the lack of a macrophage-tropic motif or signature site in the V3 region (even in purified lung macrophages), and evidence for X4 viruses in both blood and lung, are consistent with a model in which virus in the blood and lung are frequently exchanged. This is an optimistic result for patient treatment, since the presence of genotypically distinct viruses in different tissues complicates efforts to understand disease progression and optimize initial antiretroviral drug regimens.