UC Berkeley

Intracellular pathogens depend on their hosts for survival, and pathogen effectors that function at the host interface are critical to this process. Pathogens regulate their hosts through a combination of offensive and defensive strategies - capturing host machinery to carry out virulence functions while evading detection and actively inhibiting the host immune response. Here I report important advances resulting from studies of two relationships, including the oomycete Hyaloperonospora arabidopsidis (Hpa), which infects the model plant Arabidopsis thaliana, and the retrovirus HIV-1, which infects humans.

Hpa secretes a family of virulence effectors into Arabidopsis cells where they interact with host targets. Activation of the plant immune response against Hpa requires physical recognition of effectors by host Resistance proteins (R-proteins), and Hpa escapes host detection through genetic variation of effector recognition surfaces. To understand the basis for RPP1 recognition, I determined the 2.3-Å resolution crystal structure of one Hpa effector, Arabidopsis thaliana Recognized 1 (ATR1), which is recognized by the Arabidopsis R-protein Recognition of Peronospora Parasitica 1 (RPP1). We found that RPP1 recognizes distributed surfaces of ATR1, and different alleles of RPP1 recognize distinct surfaces of the effector. ATR1 belongs to an ancient family of conserved oomycete effectors that evolves rapidly through surface polymorphisms to escape host recognition while maintaining a conserved structural core.

To explore the mechanisms Human immunodeficiency virus (HIV) pathogenesis, I focused on the HIV accessory protein, Tat, which hijacks host machinery to stimulate viral transcription. HIV Tat recruits human positive transcription elongation factor b (P-TEFb) to the HIV LTR, and this recruitment is required for elongation of viral transcripts. Using a proteomics-based approach, we discovered that P-TEFb associates with several new components, including AFF4, ELL2, and homologs ENL and AF9, that form a larger Super Elongation Complex (SEC). Through in vitro reconstitution using purified recombinant proteins, I showed that AFF4 is the major scaffold that directly binds other subunits via short interaction domains. Using limited proteolysis and binding assays to structurally characterize the SEC, I found that AFF4 is an extensively disordered scaffold that remains flexible even upon binding to SEC components. Short, hydrophobic peptides on an otherwise hydrophilic, serine-rich landscape form the binding domains on AFF4. Moreover, ELL2 and ENL function as bridging components that link the SEC to another transcriptional regulator, the PAF complex (PAFc). This work establishes the overall architecture of the SEC and provides insight into how Tat, by binding P-TEFb, can recruit a large ensemble of scaffolded transcriptional regulators to stimulate viral transcription.

Together, these studies provide new insights into pathogenic mechanisms. By characterizing the structural underpinnings of interactions at two different host-pathogen interfaces, we better understand how one effector, Hpa ATR1, escapes host detection and how another effector, HIV-1 Tat, finds and recruits multiple host factors to serve a virulence function.