Faculty Bibliography

The progressive depletion of CD4 T cells underlies clinical progression to AIDS in untreated HIV-infected subjects. Most dying CD4 T cells correspond to resting nonpermissive cells residing in lymphoid tissues. Death is due to an innate immune response against the incomplete cytosolic viral DNA intermediates accumulating in these cells. The viral DNA is detected by the IFI16 sensor, leading to inflammasome assembly, caspase-1 activation, and the induction of pyroptosis, a highly inflammatory form of programmed cell death. We now show that cell-to-cell transmission of HIV is obligatorily required for activation of this death pathway. Cell-free HIV-1 virions, even when added in large quantities, fail to activate pyroptosis. These findings underscore the infected CD4 T cells as the major killing units promoting progression to AIDS and highlight a previously unappreciated role for the virological synapse in HIV pathogenesis.

BACKGROUND: Genomic integration, an obligate step in the HIV-1 replication cycle, is blocked by the integrase inhibitor raltegravir. A consequence is an excess of unintegrated viral DNA genomes, which undergo intramolecular ligation and accumulate as 2-LTR circles. These circularized genomes are also reliably observed in vivo in the absence of antiviral therapy and they persist in non-dividing cells. However, they have long been considered as dead-end products that are not precursors to integration and further viral propagation. RESULTS: Here, we show that raltegravir action is reversible and that unintegrated viral DNA is integrated in the host cell genome after raltegravir removal leading to HIV-1 replication. Using quantitative PCR approach, we analyzed the consequences of reversing prolonged raltegravir-induced integration blocks. We observed, after RAL removal, a decrease of 2-LTR circles and a transient increase of linear DNA that is subsequently integrated in the host cell genome and fuel new cycles of viral replication. CONCLUSIONS: Our data highly suggest that 2-LTR circles can be used as a reserve supply of genomes for proviral integration highlighting their potential role in the overall HIV-1 replication cycle.

Most HIV-1 replication occurs in secondary lymphoid tissues in T cells within B cell follicles. Mechanisms underlying the accumulation of HIV-1-producing cells at these sites are not understood. Antiapoptotic proteins such as Bcl-2 could promote follicular CD4(+) T cell survival, contributing to sustained virus production. Tonsils obtained from subjects without known HIV infection were disaggregated and analyzed for Bcl-2 expression in follicular (CXCR5(+)) and extrafollicular (CXCR5(-)) CD3(+)CD4(+) cells by flow cytometry. Additional tonsil cells were cultured with phytohemagglutinin (PHA) and interleukin-2 (IL-2) for 2 days, infected with either CCR5(R5) or CXCR4-tropic (X4) GFP reporter viruses, and analyzed for Bcl-2 expression. In freshly disaggregated CD3(+)CD4(+) tonsil cells, mean florescence intensity (MFI) for Bcl-2 was higher in CXCR5(+) (median, 292) compared to CXCR5(-) cells (median, 194; p=0.001). Following in vitro stimulation with PHA and IL-2, Bcl-2 MFI was higher in both CXCR5(+) cells (median, 757; p=0.03) and CXCR5(-) cells (median, 884; p=0.002) in uninfected cultures compared to freshly isolated tonsil cells. Bcl-2 MFI was higher in GFP(+)CD3(+)CD8(-) R5-producing cells (median, 554) than in X4-producing cells (median, 393; p=0.02). Bcl-2 MFI was higher in R5-producing CXCR5(+) cells (median, 840) compared to all other subsets including R5-producing CXCR5(-) cells (median, 524; p=0.04), X4-producing CXCR5(+) cells (median, 401; p=0.02), and X4-producing CXCR5(-) cells (median, 332; p=0.008). Bcl-2 expression is elevated in R5 HIV-1-producing CXCR5(+) T cells in vitro, which may contribute to propagation of R5 virus in B cell follicles in vivo.

Recent experimental data indicate that HIV-1 DNA that fails to integrate (from now on called uDNA) can by itself successfully produce infectious offspring virions in resting T cells that become activated after infection. This scenario is likely important at the initial stages of the infection. We use mathematical models to calculate the relative contribution of unintegrated and integrated viral DNA to the basic reproductive ratio of the virus, R0, and the models are parameterized with preliminary data. This is done in the context of both free virus spread and transmission of the virus through virological synapses. For free virus transmission, we find that under preliminary parameter estimates, uDNA might contribute about 20% to the total R0. This requires that a single copy of uDNA can successfully replicate. If the presence of more than one uDNA copy is required for replication, uDNA does not contribute to R0. For synaptic transmission, uDNA can contribute to R0 regardless of the number of uDNA copies required for replication. The larger the number of viruses that are successfully transmitted per synapse, however, the lower the contribution of uDNA to R0 because this increases the chances that at least one virus integrates. Using available parameter values, uDNA can maximally contribute 20% to R0 in this case. We argue that the contribution of uDNA to virus reproduction might also be important for continued low level replication of HIV-1 in the presence of integrase inhibitor therapy. Assuming a 20% contribution of uDNA to the overall R0, our calculations suggest that R0=1.6 in the absence of virus integration. While these are rough estimates based on preliminary data that are currently available, this analysis provides a framework for future experimental work which should directly measure key parameters.

The dynamics of viral infections have been investigated extensively, often with a combination of experimental and mathematical approaches. Mathematical descriptions of virus spread through cell populations are well-established in the literature, and have yielded important insights. Yet, the formulation of certain fundamental aspects of virus dynamics models remains uncertain and untested. Here, we investigate the process of infection, and in particular, the effect of varying the target cell population size on the number of productively infected cells generated. Using an in vitro single-round HIV-1 infection system, we find that the established modeling framework cannot accurately fit the data. If the model is fit to data with the lowest number of cells and is used to predict data generated with larger cell populations, the model significantly overestimates the number of productively infected cells generated. Interestingly, this deviation becomes stronger under experimental conditions that promote mixing of cells and viruses. The reason for the deviation is that the standard model makes certain over-simplifying assumptions about the fate of viruses that fail to find a cell in their immediate proximity. We derive a different model from stochastic processes that assumes simultaneous access of the virus to multiple target cells. In this scenario, if no cell is available to the virus at its location, it has a chance to interact with other cells, a process that can be promoted by mixing of the populations. This model can accurately fit the experimental data, and suggests a new interpretation of mass-action in virus dynamics models. Importance: Understanding the principles of virus growth through cell populations is of fundamental importance to virology. It helps us make informed decisions about intervention strategies aimed at preventing virus growth, such as drug treatment or vaccination approaches e.g. in HIV infection. Yet, considerable uncertainty remains in this respect. An important variable in this context is the number of susceptible cells available for virus replication. How does the number of susceptible cells influence the growth potential of the virus? Besides the importance of such information for clinical responses, a thorough understanding of this is also important for the prediction of virus levels in patients and the estimation of crucial patient parameters through the use of mathematical models. This paper investigates the relationship between target cell availability and the virus growth potential with a combination of experimental and mathematical approaches, and provides significant new insights.

HIV-1 hijacks and disrupts many processes in the cells it infects in order to suppress antiviral immunity and to facilitate its replication. Resting CD4 T cells are important early targets of HIV-1 infection in which HIV-1 must overcome intrinsic barriers to viral replication. Although resting CD4 T cells are refractory to infection in vitro, local environmental factors within lymphoid and mucosal tissues such as cytokines facilitate viral replication while maintaining the resting state. These factors can be utilized in vitro to study HIV-1 replication in resting CD4 T cells. In vivo, the migration of resting naive and central memory T cells into lymphoid tissues is dependent upon expression of CD62L (L-selectin), a receptor that is subsequently down-modulated following T cell activation. CD62L gene transcription is maintained in resting T cells by Foxo1 and KLF2, transcription factors that maintain T cell quiescence and which regulate additional cellular processes including survival, migration, and differentiation. Here we report that HIV-1 down-modulates CD62L in productively infected naive and memory resting CD4 T cells while suppressing Foxo1 activity and the expression of KLF2 mRNA. Partial T cell activation was further evident as an increase in CD69 expression. Several other Foxo1- and KLF2-regulated mRNA were increased or decreased in productively infected CD4 T cells, including IL-7ralpha, Myc, CCR5, Fam65b, S1P1 (EDG1), CD52, Cyclin D2 and p21CIP1, indicating a profound reprogramming of these cells. The Foxo1 inhibitor AS1842856 accelerated de novo viral gene expression and the sequella of infection, supporting the notion that HIV-1 suppression of Foxo1 activity may be a strategy to promote replication in resting CD4 T cells. As Foxo1 is an investigative cancer therapy target, the development of Foxo1 interventions may assist the quest to specifically suppress or activate HIV-1 replication in vivo.

While exploring the effects of aerosol IFN-gamma treatment in HIV-1/tuberculosis co-infected patients, we observed A to G mutations in HIV-1 envelope sequences derived from bronchoalveolar lavage (BAL) of aerosol IFN-gamma-treated patients and induction of adenosine deaminase acting on RNA 1 (ADAR1) in the BAL cells. IFN-gamma induced ADAR1 expression in monocyte-derived macrophages (MDM) but not T cells. ADAR1 siRNA knockdown induced HIV-1 expression in BAL cells of four HIV-1 infected patients on antiretroviral therapy. Similar results were obtained in MDM that were HIV-1 infected in vitro. Over-expression of ADAR1 in transformed macrophages inhibited HIV-1 viral replication but not viral transcription measured by nuclear run-on, suggesting that ADAR1 acts post-transcriptionally. The A to G hyper-mutation pattern observed in ADAR1 over-expressing cells in vitro was similar to that found in the lungs of HIV-1 infected patients treated with aerosol IFN-gamma suggesting the model accurately represented alveolar macrophages. Together, these results indicate that ADAR1 restricts HIV-1 replication post-transcriptionally in macrophages harboring HIV-1 provirus. ADAR1 may therefore contribute to viral latency in macrophages.

Integration is a central event in the replication of retroviruses, yet >/=90% of HIV-1 fail to integrate, resulting in accumulation of unintegrated viral DNA in cells. However, understanding what role, if any, unintegrated viral DNA plays in the natural history of HIV-1 has remained elusive. Unintegrated HIV-1 DNA is reported to possess a limited capacity for gene expression restricted to early gene products and is considered a replicative dead-end. Although the majority of peripheral blood CD4+ T cells are refractory to infection, non-activated CD4 T cells present in lymphoid and mucosal tissues are major targets for infection. Cytokines IL-2, IL-4, IL-7 and IL-15 render CD4+ T cell permissive to HIV-1 infection in the absence of cell activation and proliferation and provide a useful model for infection of resting CD4+ T cells. We find that infection of cytokine treated resting CD4+ T cells in the presence of raltegravir or with Integrase active site mutant HIV-1 yields de novo virus production following subsequent T cell activation. Infection with integration-competent HIV-1 naturally generated a population of cells generating virus from unintegrated DNA. Latent infection persists for several weeks and could be activated to virus production by a combination of a histone deacetylase inhibitor and a protein kinase C activator, or by T cell activation. HIV-1 Vpr was essential for unintegrated HIV-1 gene expression and de novo virus production in this system. Bypassing integration by this mechanism may allow the preservation of genetic information that otherwise would be lost.

Human immunodeficiency virus can spread through target cells by transmission of cell-free virus or directly from cell-to-cell via formation of virological synapses. Although cell-to-cell transmission has been described as much more efficient than cell-free infection, the relative contribution of the two transmission pathways to virus growth during multiple rounds of replication remains poorly defined. Here, we fit a mathematical model to previously published and newly generated in vitro data, and determine that free-virus and synaptic transmission contribute approximately equally to the growth of the virus population.