Virus replication is a stochastic process at a delicate equilibrium between many intrinsic and extrinsic factors. On the one hand, a single copy of viral genomic material can produce thousands of copies of progenies. The descendants consist of a simplistic molecular machinery that can rapidly spread to new environments and infect new hosts. On the other hand, viral replication and assembly is an error prone process that produces a large fraction of defective particles. The high error rate of virus replication and the huge fluctuation of the population size make it difficult to track and study the life history of viruses. In this thesis, I will introduce two powerful platforms that can accurately trace the viral life history and their applications. The genetic barcode labels each individual viral clone with a unique piece of genetic information. Coupling with the next generation sequencing, the evolutionary history of every viral lineage can be reconstructed. Secondly, the fitness profiling platform combines high throughput mutagenesis with high throughput sequencing to quantify the frequency change of mutants after various screening conditions. It efficiently measures the mutational effects on various viral systems. I will first apply the fitness profiling platform to measure the mutational effect of drug resistant mutations on HIV-1 replication. We found the pervasive positive genetic interactions between the drug resistant mutations, indicating a potential mechanism that HIV-1 maintains drug resistance. It uses multiple mutations to compensate for the fitness cost of drug resistance. Next, I modified the fitness profiling platform to quantify the mutational effect on the SARS-CoV-2 nucleocapsid protein. We measured the stability of ~8000 mutant proteins and found a continuous increasing trend of protein stability after the virus first transmitted to the human species. Thirdly, I introduced the construction of the HIV-1 barcode library and its application in single cell multi-omics. We developed a new sequencing method that can simultaneously sequence the integration site of HIV-1 and the alternative splicing forms of the viral mRNA. The barcoded HIV-1 library helps illustrate how viral integration affects virus transcription and splicing. Then I applied the barcoded HIV-1 library in humanized mice and used it to trace the clonal expansion of latently infected T cells. We designed a sequencing pipeline that can quantify the re-seeding events of each viral barcode and the clonal expansion events of each infected T cell clone. Combined with viral RNA barcode amplicon sequencing, we found the genomic features leading to less viral transcription and more T cell clonal expansion. Lastly, I presented the technical improvement that enabled accurate sequencing of the genetic barcodes and viral mutants. We benchmarked error-free sequencing methods for the next generation sequencing. I also introduced a mathematical framework for estimating the mutational effect from high throughput fitness profiling data.

Multilineage hematopoiesis suffers in the presence of HIV infection. While the hallmark of HIV infection is the massive loss of CD4+ T-cells, a number of studies have shown that the development of all blood phenotypes is disturbed through the course of infection. However, antiretroviral drugs, opportunistic infections, and the drugs used to treat them all impair hematopoiesis and confound the study of the mechanism of hematopoietic impairment in HIV-infected patient samples. These factors underscore the need to develop controlled in vitro and in vivo models to study the impact of HIV infection in the bone marrow. Here we present data that demonstrate intermediate hematopoietic progenitors are susceptible to infection both in vitro and in vivo and direct infection in part impairs hematopoietic lineage development. Further, these results were supported using the BLT humanized mouse model thus demonstrating its utility as a model to study HIV associated bone marrow pathogenesis. Innate HIV restriction factors are popular targets for manipulating infection from hematopoietic stem cells. Of the known HIV restriction factors, APOBEC3G(A3G) is unique in that it is expressed in early hematopoiesis and whose expression fluctuates significantly throughout the process of hematopoiesis. When A3G is knocked down by siRNA in hematopoietic progenitors, hematopoietic lineage commitment is clearly skewed. Erythroid and megakaryocyte lineages are impaired while granulocyte and macrophage lineages are enriched. This phenotype is maintained in a humanized mouse model. When human hematopoietic progenitors are isolated from the bone marrow of BLT mice that are reconstituted with stable A3G knockdown vectors, they show a similar skewing of hematopoietic lineage commitment. Microarray analysis of A3G knockdown in the common myeloid progenitor confirms that a number of hematopoiesis-specific genes are dysregulated when A3G expression is diminished. Further analysis suggests A3G may be exerting its effects through the cellular mRNA editing machinery.

Human immunodeficiency virus (HIV) remains a significant global health challenge, despite advancements in treatment. Antiretroviral therapy (ART) suppresses viral replication, but is not a cure. HIV persists in reservoirs of long-lived, memory CD4+ T cells harboring integrated, replication-competent provirus in a latent state. Viral persistence not only jeopardizes long-term health outcomes, but also complicates efforts to eradicate the virus, underscoring the need for innovative therapeutic approaches to achieve a cure.

One strategy known as the “kick and kill” approach aims to eliminate latently infected cells via pharmacological latency reversing agents (LRAs), which activate viral transcription, protein expression and virion production, thereby triggering these cells to be eliminated by viral-mediated cytolysis, immune detection or HIV-specific antiviral therapies. Despite some success in viral reactivation, existing LRAs failed to clear reactivated cells in vivo, emphasizing the urgent need for more effective treatments to promote the turnover of latently-infected cells.In collaboration with Paul Wender’s group at Stanford, our lab previously demonstrated that the latency-reversing capabilities and tolerability of naturally occurring protein kinase C (PKC) modulators, including bryostatin-1 and prostratin, can be improved by designing synthetic analogs of these compounds. SUW133, a potent bryostatin-1 analog, triggered latent HIV expression and led to the death of reactivated cells in humanized mice. However, whether this translates into clinically relevant outcomes remains unknown.

In this dissertation, I present a study evaluating the efficacy of PKC modulating HIV latency reversing agents, particularly SUW133, in the context of a “kick and kill” approach, as a novel therapeutic strategy against HIV persistence. Using a genetically barcoded HIV library in the context of a humanized mouse model, we quantified changes in the viral reservoir and found that SUW133 delayed viral rebound and reduced the number of unique barcoded viruses rebounding from the reservoir. Additionally, the combination of SUW133 plus NK cells further delayed rebound and reduced rebounding viral clones, even seemingly eliminating the viral reservoir in a subset of mice.

Next, we characterized the effects of SUW133 on NK cells to evaluate whether it modulates effector functions to enhance clearance of reactivated cells. While SUW133 induced changes in the activation profile of NK cells, it did not result in enhanced killing. Transcriptomic analysis revealed that CD4+ T cells had stronger responses to LRA than did NK cells, suggesting its effect on reservoir reduction is primarily due to augmenting the “kick” rather than “kill” arm of this therapeutic approach.

Lastly, I conclude with potential future directions, including some preliminary work evaluating synergistic combinations of SUW133 and histone deacetylase inhibitors (HDACi) as another novel therapeutic strategy towards latency reversal. Together, the work presented in this dissertation represents a multifaceted examination of the effects of SUW133 on latency reversal, highlighting new and exciting opportunities for its use towards an HIV cure.