Gianguido C. Cianci

Human cervicovaginal mucus contains an activity that hinders HIV-1 movement

Cervical and vaginal epithelia are primary barriers against HIV type I (HIV-1) entry during male-to-female transmission. Cervical mucus (CM) is produced by the endocervix and forms a layer locally as well as in the vaginal compartment in the form of cervicovaginal mucus (CVM). To study the potential barrier function of each mucus type during HIV-1 transmission, we quantified HIV-1 mobility in CM and CVM ex vivo using fluorescent microscopy. Virions and 200-nm PEGylated beads were digitally tracked and mean-squared displacement was calculated. The mobility of beads increased significantly in CVM compared with CM, consistent with the known decreased mucin concentration of CVM. Unexpectedly, HIV-1 diffusion was significantly hindered in the same CVM samples in which bead diffusion was unhindered. Inhibition of virus transport was envelope-independent. Our results reveal a previously unknown activity in CVM that is capable of impeding HIV-1 mobility to enhance mucosal barrier function.

Dynamics and structure of an aging binary colloidal glass

We study aging in a colloidal suspension consisting of micron-sized particles in a liquid. This system is made glassy by increasing the particle concentration. We observe samples composed of particles of two sizes, with a size ratio of 1:2.1 and a volume fraction ratio 1:6, using fast laser scanning confocal microscopy. This technique yields real-time, three-dimensional movies deep inside the colloidal glass. Specifically, we look at how the size, motion, and structural organization of the particles relate to the overall aging of the glass. Particles move in spatially heterogeneous cooperative groups. These mobile regions tend to be richer in small particles, and these small particles facilitate the motion of nearby particles of both sizes.

Correlations of structure and dynamics in an aging colloidal glass

We study concentrated colloidal suspensions, a model system which has a glass transition. Samples in the glassy state show aging, in that the motion of the colloidal particles slows as the sample ages from an initial state. We study the relationship between the static structure and the slowing dynamics, using confocal microscopy to follow the three-dimensional motion of the particles. The structure is quantified by considering tetrahedra formed by quadruplets of neighboring particles. We find that while the sample clearly slows down during aging, the static properties as measured by tetrahedral quantities do not vary. However, a weak correlation between tetrahedron shape and mobility is observed, suggesting that the structure facilitates the motion responsible for the sample aging.

Recruitment and Dynamics of Proteasome Association with rhTRIM5α Cytoplasmic Complexes During HIV‐1 Infection

A variety of proteins have been identified that restrict infection by different viruses. One such restriction factor is the rhesus macaque variant of TRIM5α (rhTRIM5α), which potently blocks infection by HIV‐1. The block to infection mediated by rhTRIM5α occurs early after entry into the host cell, generally prior to reverse transcription. However, proteasome inhibitors reveal an intermediate step of restriction in which virus can complete reverse transcription, but still fails to infect the cell. While proteasome inhibitors have been a useful tool in understanding how restriction takes place, the role of the proteasome itself during restriction has not yet been examined. Here, we characterize the interaction of rhTRIM5α and incoming virions with the proteasome. We show that proteasomes localize to rhTRIM5α cytoplasmic bodies, and this localization is more evident when the activity of the proteasome is inhibited pharmacologically. We also show that restricted virus associates with complexes of proteasomes and rhTRIM5α, suggesting that rhTRIM5α utilizes the proteasome during restriction. Finally, live cell imaging experiments reveal that virus associates with proteasomes, and proteasome inhibition affects the duration of association. Taken together, these studies implicate the proteasome as playing a functional role during rhTRIM5α restriction of incoming virions.

CCR5 Conformations Are Dynamic and Modulated by Localization, Trafficking and G Protein Association

CCR5 acts as the principal coreceptor during HIV-1 transmission and early stages of infection. Efficient HIV-1 entry requires a series of processes, many dependent on the conformational state of both viral envelope protein and cellular receptor. Monoclonal antibodies (MAbs) are able to identify different CCR5 conformations, allowing for their use as probes to distinguish CCR5 populations. Not all CCR5 MAbs are able to reduce HIV-1 infection, suggesting the use of select CCR5 populations for entry. In the U87.CD4.CCR5-GFP cell line, we used such HIV-1-restricting MAbs to probe the relation between localization, trafficking and G protein association for individual CCR5 conformations. We find that CCR5 conformations not only exhibit different localization and abundance patterns throughout the cell, but that they also display distinct sensitivities to endocytosis inhibition. Using chemokine analogs that vary in their HIV-1 inhibitory mechanisms, we also illustrate that responses to ligand engagement are conformation-specific. Additionally, we provide supporting evidence for the select sensitivity of conformations to G protein association. Characterizing the link between the function and dynamics of CCR5 populations has implications for understanding their selective targeting by HIV-1 and for the development of inhibitors that will block CCR5 utilization by the virus.

No difference in keratin thickness between inner and outer foreskins from elective male circumcisions in Rakai, Uganda.

It has been hypothesized that increased HIV acquisition in uncircumcised men may relate to a more thinly keratinized inner foreskin. However, published data are contradictory and potentially confounded by medical indications for circumcision. We tested the hypothesis that the inner foreskin was more thinly keratinized than the outer foreskin using tissues from 19 healthy, HIV-uninfected men undergoing routine prophylactic circumcision in Rakai, Uganda. Sections from 3 foreskin anatomic sites (inner, outer, and frenar band) were snap-frozen separately. Two independent laboratories each separately stained, imaged, and measured keratin thicknesses in a blinded fashion. There was no significant difference in keratin thickness between the inner (mean = 14.67±7.48 µm) and outer (mean = 13.30±8.49 µm) foreskin, or between the inner foreskin and the frenar band (mean = 16.91±12.42 µm). While the frenar band showed the greatest intra-individual heterogeneity in keratin thickness, there was substantial inter-individual variation seen in all regions. Measurements made by the two laboratories showed high correlation (r = 0.741, 95% CI, 0.533–0.864). We conclude that, despite inter- and intra-individual variability, keratin thickness was similar in the inner and outer foreskin of healthy Ugandan men, and that reduced keratin thickness is not likely to make the inner foreskin more susceptible to HIV acquisition.

Dynamic electrophoretic fingerprinting of the HIV-1 envelope glycoprotein

BackgroundInteractions between the HIV-1 envelope glycoprotein (Env) and its primary receptor CD4 are influenced by the physiological setting in which these events take place. In this study, we explored the surface chemistry of HIV-1 Env constructs at a range of pH and salinities relevant to mucosal and systemic compartments through electrophoretic mobility (EM) measurements. Sexual transmission events provide a more acidic environment for HIV-1 compared to dissemination and spread of infection occurring in blood or lymph node. We hypothesize functional, trimeric Env behaves differently than monomeric forms.ResultsThe dynamic electrophoretic fingerprint of trimeric gp140 revealed a change in EM from strongly negative to strongly positive as pH increased from that of the lower female genital tract (pHx) to that of the blood (pHy). Similar findings were observed using a trimeric influenza Haemagglutinin (HA) glycoprotein, indicating that this may be a general attribute of trimeric viral envelope glycoproteins. These findings were supported by computationally modeling the surface charge of various gp120 and HA crystal structures. To identify the behavior of the infectious agent and its target cells, EM measurements were made on purified whole HIV-1 virions and primary T-lymphocytes. Viral particles had a largely negative surface charge, and lacked the regions of positivity near neutral pH that were observed with trimeric Env. T cells changed their surface chemistry as a function of activation state, becoming more negative over a wider range of pH after activation. Soluble recombinant CD4 (sCD4) was found to be positively charged under a wide range of conditions. Binding studies between sCD4 and gp140 show that the affinity of CD4-gp140 interactions depends on pH.ConclusionsTaken together, these findings allow a more complete model of the electrochemical forces involved in HIV-1 Env functionality. These results indicate that the influence of the localized environment on the interactions of HIV with target cells are more pronounced than previously appreciated. There is differential chemistry of trimeric, but not monomeric, Env under conditions which mimic the mucosa compared to those found systemically. This should be taken into consideration during design of immunogens which targets virus at mucosal portals of entry.

Development of an imaging based virus aggregation assay for vaccine development

Background Vaccination strategies capable of eliciting neutralizing antibody responses to HIV remain elusive despite extensive efforts. Alternative antibody functions offer opportunities for protection without necessarily achieving broad neutralization breadth. Viral immune exclusion through aggregation has been proposed as an alternative protection pathway, but mechanisms for studying this phenomenon at the scale necessary for clinical trials have not been explored.

Nuclear trafficking of hiv-1 pre-integration complexes in living cells

HIV-1 viral particles engineered to incorporate integrase fused to EGFP (HIV-IN-EGFP) have proven effective to study pre-integration complexes (PICs) within nuclei of infected cells [1]. In this study we now report the live imaging analysis of nuclear PICs obtained by time-lapse microscopy. By monitoring IN-EGFP labeled complex movements within nuclei of infected cells we have been able to follow their trajectories in three dimensions. We observed PICs moving within the nuclear compartment, as well as crossing the nuclear envelope from the outer nuclear volume. Movement within the nucleus was observed primarily within regions of decondensed chromatin. To characterize these movements we calculated the mean squared displacement (MSD) of 5-minute long segment of intranuclear trajectories. In summary we obtained results indicating that 75.2% of PICs displayed active-type movement at some point during the 30 minutes of acquisition, while the remaining 24.8% exhibited only free or constrained diffusion. To gain insights into the underlying mechanism of the active transport of the IN-EGFP labeled complexes in the nucleus we tested if the active movements transport observed was mediated by actin filaments. To this end, HIV-1 trafficking was analyzed in cells treated with a drug inhibiting actin polymerization (latrunculin B). Preliminary studies revealed that treated cells showed a decrease in the number of PICs moving by active transport (47%), indicating that disruption of actin function impairs the active nuclear transport of HIV PICs. In conclusion, here we report a first detailed kinetic analysis of PICs trafficking inside the nuclear compartment. The results show that the movement of HIV PICs can take place by active transport. Because it is known that HIV shows a preference for integration into active genes these observations suggest that HIV utilizes an actin based transport mechanism to actively seek these preferred sites of integration. We believe that the identification of nuclear factors involved in HIV PIC trafficking will shed light on still unexplained aspects of HIV-1 nuclear biology.

Invariance of Structure in an Aging Colloidal Glass

We study concentrated colloidal suspensions, a model system which has a glass transition. The non‐equilibrium nature of the glassy state is most clearly highlighted by aging — the dependence of the system’s properties on the time elapsed since vitrification. Fast laser scanning confocal microscopy allows us to image a colloidal glass and track the particles in three dimensions. We analyze the static structure in terms of tetrahedral packing. We find that while the aging of the suspension clearly affects its dynamics, none of the geometrical quantities associated with tetrahedra change with age.