Mechanisms of innate immune activation by human immunodeficiency virus type 1
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Abstract
Human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS), has claimed tens of millions of lives since the emergence of the global HIV-1 pandemic in the 1980s. Chronic HIV-1 infection leads to gradual loss of CD4+ T cells in most people with HIV (PWH), ultimately leading to profound immune collapse known as AIDS and fatal opportunistic infections. Combined antiretroviral therapy (ART) involves treating PWH with a cocktail of antiretroviral inhibitors that target multiple stages of the viral replication cycle. In most individuals, this treatment achieves durable remission of viral replication and results in an undetectable viral load in peripheral blood. Although ART arrests progression to AIDS and onward viral transmission, a large reservoir of latently- and chronically-infected cells remains in essentially all tissue sites, and viral rebound usually occurs upon cessation of ART. As intracellular parasites, viruses must use sophisticated mechanisms to produce and assemble viral components while suppressing activation of cell-intrinsic innate immune effectors. In Chapter One, I report that coordination of HIV-1 assembly by the viral polyprotein Gag suppresses inappropriately-timed protease (PR) activity to avoid inflammasome activation. Employing mutants of Gag, I show that disruption of domains controlling the viral assembly site (matrix, MA) or virus particle release (nucleocapsid, NC and p6) lead to premature activation of PR, which is recognized by the inflammasome sensor caspase recruitment domain family member 8 (CARD8), resulting in interleukin 1β (IL-1β) secretion and pyroptotic cell death. The generation of active PR required myristoylation of Gag and capsid-capsid interactions which drive Gag aggregation, though Gag myristoylation was not necessary if PR activation was induced pharmacologically. Together, these data support a model where dysregulated viral assembly generates active soluble PR which can be sensed at a membrane-proximal or membrane-distal cytoplasmic site. Further, I demonstrate that previously observed host-adaptive mutations in HIV-1 MA (M30K) and p6 (PTAP motif duplication) associated with greater fitness in humans improve infected CD4+ T cell survival in a PR-dependent manner. Altogether, the work described in Chapter One reveals virus-encoded mechanistic control over PR activation and CARD8 sensing during assembly by HIV-1 Gag and suggests that assembly-regulated CARD8 activation may influence the trajectory of HIV-1 evolution and fitness in humans.
Heterogenous transcription start site (TSS) usage by the integrated HIV-1 provirus dictates the structure and function of unspliced HIV-1 RNA (usRNA). Previous reports indicate that the expression and Rev/CRM1-mediated nuclear export of HIV-1 usRNA in macrophages activate melanoma differentiation-associated protein 5 (MDA5), mitochondrial antiviral-signaling protein (MAVS), and innate immune signaling cascades. In Chapter Two, I report that MDA5 sensing of viral usRNA is strictly determined by TSS, 5’ leader structure, and RNA function. I show that cap-sequestered HIV-1 usRNA (cap1G) destined for packaging is specifically targeted by MDA5, while translation-destined (cap3G) usRNA is remarkably immunologically silent. Using mutant viruses which express usRNA with altered 5’ cap structural conformations, or inclusion of a retroviral constitutive transport element which drives messenger RNA (mRNA)-like nuclear RNA export factor 1 (NXF1)-dependent nuclear export of viral usRNA, I show that cap exposure and nuclear export pathway choice are major determinants of both lentiviral RNA immunogenicity and function. In total, I identify innate immune evasion as a possible rationale for the universal conservation of heterogenous TSS usage among ancestral and extant HIV-1 isolates and shed light on how MDA5 fundamentally discriminates between self and non-self RNAs.
Chronic innate immune activation is a hallmark of disease progression to AIDS, and occurs during infection even with successful ART treatment, which leads to a greater incidence of negative health outcomes for PWH relative to their age-matched counterparts without HIV. Separately, innate immune activation and viral subversion of these innate responses is also relevant to mucosal transmission, a low-efficiency event where the virus must traverse a complex tissue barrier prone to robust antiviral responses and establish efficient replication in a naïve host. Together, the findings described in Chapters One and Two deepen our understanding of the innate immunology of HIV-1. These results define innate immune sensing mechanisms which target late events in the viral lifecycle (viral genome packaging and assembly) and the viral immune evasion strategies which facilitate the establishment of chronic infection. Future studies should incorporate these findings to further investigate mechanisms of chronic inflammation in PWH and inform therapeutic strategies.
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2026