The identification of viral traits that might enhance mucosal transmission is an important goal for vaccine development and other prevention strategies. A first step in characterizing such traits is the identification of T/F viruses, while a second step is the selection of appropriate chronic controls. Virological traits that are strongly associated with transmission, such as CCR5 use, should be readily identifiable when comparing T/F viruses to virtually any control group, while identifying more subtle phenotypes will greatly depend on the choice of control viruses, perhaps explaining discrepancies in genetic and phenotypic transmission signatures identified by different groups 
. Finally, the use of in vitro
assays that recapitulate key steps in mucosal transmission are needed to identify properties unique to T/F viruses. Here, we have compiled a relatively large panel of both Envs and IMCs representing subtype C T/F and chronic control viruses, and developed a series of infection assays using virus pseudotypes, replication competent viruses, cell lines and primary human CD4+ T cells to improve our ability to identify viral phenotypes associated with transmission.
Env glycoproteins of HIV-1 can differ significantly in the efficiencies with which they utilize CD4 and the viral coreceptors, which in turn can impact viral tropism 
. Given the variability in expression levels of entry cofactors on different cell subsets as well as between individuals 
, it is easy to envision several ways in which Env function could impact transmission efficiency at the level of virus entry. To date, several genetic Env signatures have been reported, with more compact variable loop structures and fewer PNGs being the most frequent findings 
. It is possible that these genetic traits impact Env function in ways that increase transmission fitness. However, to date no consistent T/F phenotype has been described. Thus, it is possible that mucosal transmission is a stochastic event where any reasonably functional R5 or dual tropic Env can initiate a productive infection 
. However, it is also possible that the in vitro
assays employed thus far have failed to reveal subtler or more transmission-specific phenotypic differences. The recent finding that some gp120 proteins from early HIV-1 infections can bind to the α4β7 integrin is consistent with this, although the ability of T/F viruses to productively interact with α4β7 was not explored.
To determine whether subtype C T/F viruses, which account for the great majority of new infections worldwide, utilize CD4 or CCR5 with enhanced efficiency, we tested both T/F and chronic Env constructs in pseudotyping assays. Consistent with previous results for subtype B 
and subtype C 
, we failed to observe differences in both CCR5 and CD4 utilization. This is in contrast to findings by Etemad and colleagues who reported enhanced CCR5 utilization by Envs from individuals with chronic subtype A infection, although only V1–V5 fragments were tested in the context of chimeric viruses 
. Similarly, Nawaz and colleagues found that gp120s from three subjects acutely infected with subtype A and C viruses bound to dodecameric but not monomeric CD4, while gp120s from subsequent time points of two of the same subjects bound to CD4 in both forms, suggesting an increase in CD4 affinity in later stages of infection 
. These results may be specific to the particular Envs 
or Env fragments 
used, or due to the fact that gp120 and particle-associated Env trimers bind CD4 differently. In either case, current data utilizing a large number of Env constructs strongly suggests that the mucosal bottleneck is not the result of selective transmission of viruses with highly efficient CD4 or CCR5 use 
, or with increased efficiency of entry into particular CD4+ T cells subsets 
We also examined whether subtype C T/F and chronic Envs differed in their interaction with the gut homing integrin α4β7 as recently proposed 
, although previous data are almost entirely based on gp120 binding studies. In many ways, the α4β7 hypothesis is an attractive one. This integrin is expressed at high levels on activated CD4+ T cells in the gut 
and cervicovaginal mucosa 
, both representing major sites of HIV replication early in infection 
. Moreover, intravenous administration of an anti-α4β7 mAb in rhesus macaques prior to and during acute infection with SIVmac239 resulted in decreased virus loads, perhaps by inhibiting trafficking of α4β7-positive T cells to the GI tract 
. Finally, gp120-induced α4β7 signaling could promote virus replication through increased cell-to-cell adhesion. However, the ability of HIV-1 gp120 to bind α4β7 is far from universal - the commonly studied subtype B gp120s examined to date either do not bind to α4β7 or do so weakly, with the exception of SF162 
. Nonetheless, several gp120 proteins derived from early subtype A or subtype C infections have been shown to exhibit α4β7 binding capacity, and there is an obvious link between some α4β7 binding properties (fewer PNGs in the V1–V4 region) and genotypes associated with virus transmission in subtype C viruses 
. While monomeric gp120 binds CD4, viral coreceptors and most broadly neutralizing antibodies, it differs from virion-associated Env trimers in important ways. Perhaps the best example is that numerous antibodies that bind to gp120 fail to neutralize the cognate Env trimer, consistent with both conformational differences and the fact that certain gp120 domains are sequestered in the oligomeric molecule. Thus, a key question that remains relatively unexplored is whether α4β7 binding by gp120 translates into an interaction by trimeric Env that influences virus infection and spread. To address this question, we concentrated on virus infection assays rather than gp120 binding experiments.
In our attempts to define the role of α4β7 in HIV-1 transmission, we were able to replicate a previous key finding, namely that saturating levels of antibodies to α4β7 modestly suppressed infection and replication by the prototypic subtype B strain HIV-1/SF162 
. The inhibitory effects of α4β7 antibodies on SF162 infection were both transitory and most evident when low levels of virus input were used, which is precisely what would be expected if α4β7 functioned as an attachment factor 
. Attachment of virus particles to the host cell surface is a significant rate-limiting step to virus infection in vitro
, but can be overcome in part by spinoculation 
, the inclusion of polycations that enhance viral binding 
, or the expression of virus attachment factors such as CD209 or CD209R 
. In the case of attachment factors, their ability to enhance infection is most pronounced when low levels of virus are used. Thus, our finding of a partial inhibition of SF162 replication in α4β7-positive T cells six days post infection at the lowest virus input is entirely consistent with previous reports and shows that our assays are sufficiently sensitive to measure the impact of α4β7 blockade on virus infection. Despite this, we found no inhibition of any T/F or chronic subtype B or C virus using cells from multiple donors and levels of virus empirically determined to be barely sufficient to establish a spreading infection. These findings are consistent with those of Pauls and colleagues, who found that a mAb to α4 used for the treatment of multiple sclerosis and Crohn's disease did not impact infection of atRA-treated CD4+ T cells by several HIV-1 strains, including two with the LDI/V tripeptide binding motif in the V2 region 
. Since most of our T/F and chronic viruses possessed the α4β7-binding tripeptide motif as well as below average numbers of PNGs in the V1/V2 region, selection bias - i.e. the preferential inclusion of viruses that would be unlikely to interact with α4β7 - can also be excluded. Thus, we favor the hypothesis that not all Envs that can bind α4β7 in the form of gp120 necessarily do so as unliganded trimers.
Our failure to detect enhancement of viral infection of human CD4+ T cells by primary subtype B or C viruses, including T/F viruses, due to α4β7 interaction is by no means definitive, but does suggest that extrapolating results from gp120 binding assays to more complex virion infectivity studies may be misleading. It is possible that α4β7 interactions will be more important in other types of infection assays. In addition, we have not tested the ability of gp120 proteins derived from our viruses to bind to α4β7, although the relevance of such findings remains uncertain unless the corresponding trimeric Env exhibits similar properties. Our results demonstrate the importance of using replication-competent viruses to study properties associated with mucosal transmission. In contrast to single-round pseudovirus assays, experiments with IMCs are unbiased with respect to the genes that could influence fitness and enable detection of subtle differences following multiple rounds of replication. Thus, T/F and chronic IMCs are ideal reagents for future studies of phenotypes that may influence HIV-1 transmission.