Hepatitis C virus (HCV) is a major public health threat, chronically infecting approximately 180 million people worldwide and leading to liver diseases such as cirrhosis and hepatocellular carcinoma. To date, efforts to generate a prophylactic or therapeutic vaccine have been unsuccessful. The failure of vaccine candidates, as well as the rapid emergence of drug-resistant variants, is almost entirely due to the hypervariable nature of HCV. Interestingly, while HCV is highly variable, recombination events between HCV strains are rare. This lack of recombination suggests that immune escape and drug resistance mutations are rarely transferred between strains, providing clear implications for treatment paradigms in patients infected with multiple HCV strains.

When HCV infects a host cell, that cell becomes resistant to further infection by HCV but not other viruses, such as dengue virus. This resistance, termed superinfection exclusion, acts to limit the co-occupancy of cells with multiple viral genomes, and thus prevents recombination. However, the mechanism of this block is poorly understood.

In this thesis I focus on further understanding why these recombination events are so rarely observed, through detailed investigations of the phenomena of superinfection exclusion and intracellular competition between HCV genomes during cell division. To further characterize HCV superinfection exclusion, we created a class of novel reporter HCV strains selected for their ability to infect cells already replicating HCV RNA. Importantly, the evolution of the ability to "superinfect" cells caused these viral strains to become more infectious generally, and led these viruses to expel the HCV RNA already present in the host cells. We further identified key changes in the HCV genome that allowed the virus to overcome the superinfection block.

In addition to our work on superinfection exclusion, we identified a novel means of intracellular competition between HCV genomes co-occupying a host cell that occurs at, or shortly following, mitosis. In the setting of cellular division, we found that when two viral genomes of equivalent fitness are present within a cell, they have an equal opportunity to exclude the other. In a population of dividing cells, the competition between viral genomes proceeds apace, randomly clearing one or the other genome from cells in the span of 9-12 days. In addition to superinfection exclusion, this intracellular competition due to mitosis is likely to further limit HCV's genetic diversity and ability to recombine in vivo.

In sum, this thesis demonstrates that viral competition in HCV infection occurs on many more levels than was previously appreciated. This competition acts to prevent coinfection of host cells and likely explains the lack of natural recombination in HCV biology. Further, these findings shed additional light on basic aspects of HCV replication and hopefully lead in the future to novel therapies for this critical disease.

Acquired Immunodeficiency virus (AIDS) has killed 36 million people since the first cases were reported in 1981, and is considered a worldwide pandemic. AIDS is caused by the human immunodeficiency virus (HIV), which first originated in Africa in the early 1900s, and by the 1980s had spread around the globe. HIV is a lentivirus that slowly destroys the immune system by infecting and killing CD4+ T cells. There is currently no cure or vaccine for HIV. However, drug therapy exists that is capable of controlling HIV infection, and prevents the progression to AIDS. Drug therapy cannot eradicate HIV because the virus can become transcriptionally silent after integrating its genetic material into the host cell's genome, resulting in latent infection. HIV latency is a reversible state, and allows the virus to remain within an infected individual for the entirety of their life-span. The study of HIV latency has been ongoing since 1986, but has been hindered by two major problems: 1) latently infected cells are extremely rare, in vivo; and 2) latently infected cells are indistinguishable from uninfected cells. The work of this dissertation focused on the development and utilization of an HIV dual-reporter virus that could identify latently infected cells, and distinguish them from productively infected and uninfected cells. The HIV dual-reporter viruses utilize two different fluorescent proteins. One fluorescent protein is under the control of the HIV LTR promoter, and identifies cells that are productively infected, while the other fluorescent protein is under the control of a constitutive promoter, independent of the viral promoter, and identifies cells that have integrated provirus. Infection of Jurkat cells and primary CD4+ T cells with these dual-reporter viruses allows for the identification and purification of latently infected cells. This isolated latent population which only expresses the fluorescent protein that is under the control of the constitutive promoter, is devoid of viral transcripts and viral proteins. One of these dual-reporter viruses, HIV Duo-Fluo I, was used to study the role of T cell activation on the establishment of HIV latency in primary CD4+ T cells. Resting primary CD4+ T cells are capable of supporting productive HIV infection, but show a propensity towards latent infection, while activated primary CD4+ T cells are capable of supporting latent infection, but show a propensity towards productive infection. Further, activated primary CD4+ T cells are capable of supporting latent infection without the need to return to a resting state. Our findings suggest that HIV latency is not completely dependent on T cell activation and that infection of both resting and activated primary CD4+ T cells can contribute to the HIV latent reservoir.

The pathway causing CD4 T-cell death in HIV-infected hosts remains poorly understood although apoptosis has been proposed as a key player. Our studies now show that caspase-3-mediated apoptosis only mediates the death of only a small fraction (<5%) of the total CD4 T cells that correspond to activated cells. The remaining >95% of quiescent lymphoid CD4 T-cells die by caspase-1-mediated pyroptosis triggered as a result of abortive viral infection occurring in nonpermissive resting CD4 T cells. Pyroptosis is an intensely inflammatory form of programmed cell death where cytoplasmic contents and pro-inflammatory cytokines including IL-1β and IL-18 are released. This death pathway thus links the two signature events in HIV infection--CD4 T-cell depletion and chronic inflammation--in a single process and creates a vicious pathogenic cycle where dying CD4 T-cells release inflammatory signals that attract more cells to die. This cycle can be broken with caspase-1 inhibitors, including a compound already shown to be safe and well-tolerated in humans. These inhibitors could form a new class of "anti-AIDS" therapeutics that target the host rather than the virus. We further show that cell-to-cell transmission of HIV is obligately required for the induction of pyroptosis. Cell-free HIV-1 virions, even when added in large quantities, due not suffice. These findings underscore the infected CD4 T cell as the major killing unit promoting progression to AIDS and highlight a previously unappreciated biological role for the virological synapse.