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Recombination has been shown to contribute to human immunodeficiency virus-1 (HIV-1) evolution in vivo, but the underlying dynamics are extremely complex, depending on the nature of the fitness landscapes and of epistatic interactions. A less well-studied determinant of recombinant evolution is the mode of virus transmission in the cell population. HIV-1 can spread by free virus transmission, resulting largely in singly infected cells, and also by direct cell-to-cell transmission, resulting in the simultaneous infection of cells with multiple viruses. We investigate the contribution of these two transmission pathways to recombinant evolution, by applying mathematical models to in vitro experimental data on the growth of fluorescent reporter viruses under static conditions (where both transmission pathways operate), and under gentle shaking conditions, where cell-to-cell transmission is largely inhibited. The parameterized mathematical models are then used to extrapolate the viral evolutionary dynamics beyond the experimental settings. Assuming a fixed basic reproductive ratio of the virus (independent of transmission pathway), we find that recombinant evolution is fastest if virus spread is driven only by cell-to-cell transmission and slows down if both transmission pathways operate. Recombinant evolution is slowest if all virus spread occurs through free virus transmission. This is due to cell-to-cell transmission 1, increasing infection multiplicity; 2, promoting the co-transmission of different virus strains from cell to cell; and 3, increasing the rate at which point mutations are generated as a result of more reverse transcription events. This study further resulted in the estimation of various parameters that characterize these evolutionary processes. For example, we estimate that during cell-to-cell transmission, an average of three viruses successfully integrated into the target cell, which can significantly raise the infection multiplicity compared to free virus transmission. In general, our study points towards the importance of infection multiplicity and cell-to-cell transmission for HIV evolution.

The infection of cells by multiple copies of a given virus can impact viral evolution in a variety of ways, yet some of the most basic evolutionary dynamics remain underexplored. Using computational models, we investigate how infection multiplicity affects the fixation probability of mutants, the rate of mutant generation, and the timing of mutant invasion. An important insight from these models is that for neutral and disadvantageous phenotypes, rare mutants initially enjoy a fitness advantage in the presence of multiple infection of cells. This arises because multiple infection allows the rare mutant to enter more target cells and to spread faster, while it does not accelerate the spread of the resident wild-type virus. The rare mutant population can increase by entry into both uninfected and wild-type-infected cells, while the established wild-type population can initially only grow through entry into uninfected cells. Following this initial advantageous phase, the dynamics are governed by drift or negative selection, respectively, and a higher multiplicity reduces the chances that mutants fix in the population. Hence, while increased infection multiplicity promotes the presence of neutral and disadvantageous mutants in the short-term, it makes it less likely in the longer term. We show how these theoretical insights can be useful for the interpretation of experimental data on virus evolution at low and high multiplicities. The dynamics explored here provide a basis for the investigation of more complex viral evolutionary processes, including recombination, reassortment, as well as complementary/inhibitory interactions.

BackgroundHIV-1 integration is prone to a high rate of failure, resulting in the accumulation of unintegrated viral genomes (uDNA) in vivo and in vitro. uDNA can be transcriptionally active, and circularized uDNA genomes are biochemically stable in non-proliferating cells. Resting, non-proliferating CD4 T cells are prime targets of HIV-1 infection and latently infected resting CD4 T cells are the major barrier to HIV cure. Our prior studies demonstrated that uDNA generates infectious virions when T cell activation follows rather than precedes infection.ResultsHere, we characterize in primary resting CD4 T cells the dynamics of integrated and unintegrated virus expression, genome persistence and sensitivity to latency reversing agents. Unintegrated HIV-1 was abundant in directly infected resting CD4 T cells. Maximal gene expression from uDNA was delayed compared with integrated HIV-1 and was less toxic, resulting in uDNA enrichment over time relative to integrated proviruses. Inhibiting integration with raltegravir shunted the generation of durable latency from integrated to unintegrated genomes. Latent uDNA was activated to de novo virus production by latency reversing agents that also activated latent integrated proviruses, including PKC activators, histone deacetylase inhibitors and P-TEFb agonists. However, uDNA responses displayed a wider dynamic range, indicating differential regulation of expression relative to integrated proviruses. Similar to what has recently been demonstrated for latent integrated proviruses, one or two applications of latency reversing agents failed to activate all latent unintegrated genomes. Unlike integrated proviruses, uDNA gene expression did not down modulate expression of HLA Class I on resting CD4 T cells. uDNA did, however, efficiently prime infected cells for killing by HIV-1-specific cytotoxic T cells.ConclusionsThese studies demonstrate that contributions by unintegrated genomes to HIV-1 gene expression, virus production, latency and immune responses are inherent properties of the direct infection of resting CD4 T cells. Experimental models of HIV-1 latency employing directly infected resting CD4 T cells should calibrate the contribution of unintegrated HIV-1.

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.

UnlabelledThe 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 oversimplifying assumptions about the fate of viruses that fail to find a cell in their immediate proximity. We derive from stochastic processes a different model 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.ImportanceUnderstanding 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.

Integration is a central event in the replication of retroviruses, yet ≥ 90% of HIV-1 reverse transcripts 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, nonactivated CD4 T cells present in lymphoid and mucosal tissues are major targets for infection. Treatment with cytokine interleukin-2 (IL-2), IL-4, IL-7, or IL-15 renders CD4(+) T cells permissive to HIV-1 infection in the absence of cell activation and proliferation and provides a useful model for infection of resting CD4(+) T cells. We found that infection of cytokine-treated resting CD4(+) T cells in the presence of raltegravir or with integrase active-site mutant HIV-1 yielded 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 persisted 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.

Abstract Background The dynamics of viral infections have been studied extensively in a variety of settings, both experimentally and with mathematical models. The majority of mathematical models assumes that only one virus can infect a given cell at a time. It is, however, clear that especially in the context of high viral load, cells can become infected with multiple copies of a virus, a process called coinfection. This has been best demonstrated experimentally for human immunodeficiency virus (HIV), although it is thought to be equally relevant for a number of other viral infections. In a previously explored mathematical model, the viral output from an infected cell does not depend on the number of viruses that reside in the cell, i.e. viral replication is limited by cellular rather than viral factors. In this case, basic virus dynamics properties are not altered by coinfection. Results Here, we explore the alternative assumption that multiply infected cells are characterized by an increased burst size and find that this can fundamentally alter model predictions. Under this scenario, establishment of infection may not be solely determined by the basic reproductive ratio of the virus, but can depend on the initial virus load. Upon infection, the virus population need not follow straight exponential growth. Instead, the exponential rate of growth can increase over time as virus load becomes larger. Moreover, the model suggests that the ability of anti-viral drugs to suppress the virus population can depend on the virus load upon initiation of therapy. This is because more coinfected cells, which produce more virus, are present at higher virus loads. Hence, the degree of drug resistance is not only determined by the viral genotype, but also by the prevalence of coinfected cells. Conclusions Our work shows how an increased burst size in multiply infected cells can alter basic infection dynamics. This forms the basis for future experimental testing of model assumptions and predictions that can distinguish between the different scenarios. Reviewers This article was reviewed by RJdeB, RMR and MK.

It is commonly held that the traditional European classics or Great Books cannot constitute the basis of an inclusive liberal education because they do not reflect the diverse cultural identities of contemporary students. This essay argues for a return to the older view, memorably expressed by W.E.B. Du Bois, that the classics are radically inclusive because they speak to a common human identity. This view is best understood in light of the ancient Greek distinction between physis (nature) and nomos (custom or law). Greek philosophic universalism has shown an ability to transcend profound cultural differences, such as those separating Alfarabi from Maimonides, or Shakespeare from Du Bois. The essay rebuts the frequently heard objections that the universalism of the classics is really a mask for white male hegemony and that it fails to do justice to the unique personal experiences of the oppressed and marginalized. The essay affirms that education must be particularistic as well as universalistic, but it argues that the universalism of the classics remains the key to the most perfect form of inclusion, namely, the friendship of those seeking the truth. [ABSTRACT FROM AUTHOR]

The human immunodeficiency virus (HIV) viral protein R (vpr) is conserved within all genomes of HIV-1 and HIV-2. Rhabdomyosarcoma cell lines were transfected with the vpr gene to determine its function in HIV pathogenesis. The results showed that vpr could induce cell growth inhibition and differentiation. These results indicate that vpr is a regulatory protein that plays a role in cell proliferation and differentiation.

Abstract Background The integration of HIV-1 DNA into cellular chromatin is required for high levels of viral gene expression and for the production of new virions. However, the majority of HIV-1 DNA remains unintegrated and is generally considered a replicative dead-end. A limited amount of early gene expression from unintegrated DNA has been reported, but viral replication does not proceed further in cells which contain only unintegrated DNA. Multiple infection of cells is common, and cells that are productively infected with an integrated provirus frequently also contain unintegrated HIV-1 DNA. Here we examine the influence of an integrated provirus on unintegrated HIV-1 DNA (uDNA). Results We employed reporter viruses and quantitative real time PCR to examine gene expression and virus replication during coinfection with integrating and non-integrating HIV-1. Most cells which contained only uDNA displayed no detected expression from fluorescent reporter genes inserted into early (Rev-independent) and late (Rev-dependent) locations in the HIV-1 genome. Coinfection with an integrated provirus resulted in a several fold increase in the number of cells displaying uDNA early gene expression and efficiently drove uDNA into late gene expression. We found that coinfection generates virions which package and deliver uDNA-derived genomes into cells; in this way uDNA completes its replication cycle by viral complementation. uDNA-derived genomes undergo recombination with the integrated provirus-derived genomes during second round infection. Conclusion This novel mode of retroviral replication allows survival of viruses which would otherwise be lost because of a failure to integrate, amplifies the effective amount of cellular coinfection, increases the replicating HIV-1 gene pool, and enhances the opportunity for diversification through errors of polymerization and recombination.

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric, mucin-like, 120-kDa glycoprotein that binds to P-, E-, and L-selectins. PSGL-1 is expressed primarily on the surface of lymphoid and myeloid cells and is up-regulated during inflammation to mediate leukocyte tethering and rolling on the surface of endothelium for migration into inflamed tissues. Although it has been reported that PSGL-1 expression inhibits HIV-1 replication, the mechanism of PSGL-1– mediated anti-HIV activity remains to be elucidated. Here we report that PSGL-1 in virions blocks the infectivity of HIV-1 particles by preventing the binding of particles to target cells. This inhibitory activity is independent of the viral glycoprotein present on the virus particle; the binding of particles bearing the HIV-1 envelope glycoprotein or vesicular stomatitis virus G glycoprotein or even lacking a viral glycoprotein is impaired by PSGL-1. Mapping studies show that the extracellular N-terminal domain of PSGL-1 is necessary for its anti–HIV-1 activity, and that the PSGL-1 cytoplasmic tail contributes to inhibition. In addition, we demonstrate that the PSGL-1–related monomeric E-selectin–binding glycoprotein CD43 also effectively blocks HIV-1 infectivity. HIV-1 infection, or expression of either Vpu or Nef, down-regulates PSGL-1 from the cell surface; expression of Vpu appears to be primarily responsible for enabling the virus to partially escape PSGL-1–mediated restriction. Finally, we show that PSGL-1 inhibits the infectivity of other viruses, such as murine leukemia virus and influenza A virus. These findings demonstrate that PSGL-1 is a broad-spectrum antiviral host factor with a unique mechanism of action. [ABSTRACT FROM AUTHOR]

Moral virtue is supposed to be its own reward. In chapters 15 and following of The Prince, Machiavelli argues forcefully that this principle is not conducive to the happiness of rulers and their subjects (which is to say, of everyone). One must learn to judge by effects and to call virtuous those actions, and only those actions, which are truly rewarding because they promote one's security, prosperity, and honor. This article shows that Aristotle--a primary, though unnamed, adversary of Machiavelli here--was familiar with arguments of this kind and replied to them. The article focuses on the two thinkers' views regarding the relation between the noble and the good, with particular attention to their contrasting presentations of the virtue of liberality. [ABSTRACT FROM AUTHOR]