CROI 2016 Abstract eBook

Abstract Listing

Oral Abstracts

58 How HIV-1 Nef Downregulation Hijacks Clathrin-Coated Vesicles James H. Hurley , Univ of California Berkeley, Berkeley, CA, USA

The lentiviruses HIV and simian immunodeficiency virus (SIV) subvert intracellular membrane traffic as part of their replication cycle. The lentiviral Nef protein helps viruses evade innate and adaptive immune defenses by hijacking the clathrin adaptor complexes AP-1 and AP-2. These in turn drive the downregulation of MHC-I, CD4, and SERINC3 and 5, among other cellular targets. We have been building up a structural picture that explains how HIV-1 Nef uses at least two different sites- the region around Asp123 and the dileucine motif- to hijack AP complexes in two different ways. In particular, HIV-1 Nef and the small G-protein Arf1 use a surprisingly complicated mechanism to hijack AP-1, which is responsible for MHC-I downregulation. Arf1 glues AP-1 into dimers and trimers that in turn form a hexagonal undercoat to clathrin. Nef promotes trimer formation and the generation of the hexagonal lattice. The mode of Nef action depends upon the presence of cargo. The action of Nef with respect to AP-1 appears to be highly regulated, and a novel Nef regulatory site involved in controlling the structure of AP-1 trimers will be described. Implications for AP-2 dependent downregulation of the SERINCs, and similarites and differences between Nef hijacking of AP-1 and AP-2 will be considered. 59 APOBEC3F-Vif Binding Interface Elucidated by Numerous Experimental Approaches Reuben S. Harris , Howard Hughes Med Inst, Minneapolis, MN, USA APOBEC3 family DNA cytosine deaminases provide overlapping defenses against pathogen infections. However, most viruses have elaborate evasion mechanisms such as the HIV-1 Vif protein, which subverts cellular CBF-beta and scaffolds the assembly of a polyubiquitin ligase complex to degrade these enzymes. Despite advances in APOBEC3 and Vif biology, a full understanding of this direct host-pathogen conflict has been elusive. We combine virus adaptation, structural, and computational studies to interrogate the APOBEC3F-Vif interface and build a robust model for this vital host-pathogen interaction. First, new structures of the Vif binding domain of APOBEC3F were solved by x-ray crystallography. Second, a recurring compensatory amino acid substitution in HIV-1 Vif from virus adaptation experiments provided an initial docking constraint, and microsecond molecular dynamics simulations were used to optimize interface contacts. Third, single-cycle and spreading virus infectivity experiments were used to validate a long-lasting electrostatic interaction predicted by molecular dynamics simulations to occur between APOBEC3F E289 and HIV-1 Vif R15. Our studies have yielded a comprehensive structural model for the APOBEC3F-Vif interaction, which explains most prior genetic data. Taken together with the dynamic nature of this host-pathogen interaction, our data suggest a “wobble model” to explain how HIV-1 Vif has evolved to bind different APOBEC3 enzymes and, more generally, explain how pathogens may evolve to escape innate host defenses ( i.e ., a molecular explanation for the Red Queen hypothesis). Our studies also suggest that the APOBEC-Vif interface may be a less favorable target for new antiviral drug development in comparison to other heterologous surfaces of the Vif/CBF-beta ubiquitin ligase complex. 60 CNS HIV Infection: Cerebrospinal Fluid and Blood Biomarkers Magnus Gisslén , Univ of Gothenburg, Gothenburg, Sweden Neurological complications are common in patients with HIV and the prevalence of neurocognitive impairment is high also among those on suppressive antiretroviral treatment (ART). Sometimes neurocognitive complications may be ascribed to CNS injury that occurred before treatment initiation (inactive disease), especially in patients with a low CD4 cell nadir; and other times to ongoing neuronal injury accompanied by chronic intrathecal immunoactivation (active disease). Cerebrospinal fluid (CSF) biomarkers of viral replication, immunoactivation, and neuronal injury provide an objective means of measuring ongoing HIV CNS infection and inflammation along with its effect on brain cells. This presentation will consider the usefulness of CSF biomarkers in measuring HIV CNS disease, particularly in HIV-infected patients on ART. Employing biomarkers for differential diagnostic purposes will also be covered. Biomarkers of CNS immunoactivation and brain injury may also be useful for research into latency and cure, and as tools for measuring the reservoir and the effect of latency-reversing agents on possible CNS immune activation and injury. However, the need to sample CSF limits the application of those measurements in a number of clinical and scientific contexts. A sensitive blood biomarker of neuronal injury continues to be sought. The resent development of a novel ultrasensitive Single Molecule Array (Simoa) immunoassay for the quantification of the neurofilament light chain protein (NFL) in blood will be presented. 61 Neuroimaging of HIV in the Brain Beau M. Ances , Washington Univ in St. Louis, St. Louis, MO, USA HIV enters the brain soon after seroconversion and can cause HIV associated neurocognitive disorders (HAND). While the more severe and progressive forms of HAND are now less prevalent due to combination antiretroviral therapy (cART), ~ 40% of HIV-infected (HIV+) patients continue to have cognitive impairment. Some HIV+ individuals who have effective plasma HIV-1 RNA suppression with cART still develop HAND due HIV reservoirs in the central nervous system. It is often difficult to diagnose HAND in the outpatient setting as detailed neuropsychological performance testing is required. Additional biomarkers that are relatively easy to obtain and are clinically relevant are needed for assessing HIV associated immune activation. Recently developed non-invasive magnetic resonance imaging (MRI) techniques have great potential to serve as biomarkers. This talk will review the application of advanced neuroimaging techniques [e.g. volumetric MRI, diffusion tensor imaging (DTI), functional MRI (fMRI)] in HIV+ individuals. Each neuroimaging methods can offer unique insight into mechanisms underlying neuroHIV, could monitor disease progression, and may assist in evaluating the efficacy of cART regimens in the CNS. It is hoped that continued development of neuroimaging methods will allow them to be easily incorporated across multiple sites and included in future HAND guidelines. 62 CNS as an HIV Reservoir Ronald I. Swanstrom , Univ of North Carolina at Chapel Hill, Chapel Hill, NC, USA Replication of HIV-1 within the CNS compartment can only be bad. At a minimum the detection by the immune system of replicating virus within the CNS leads to a neuro-toxic inflammatory response. If the clearance of virus is incomplete then a persistent inflammatory response could provide the basis for sustained cell damage within the CNS. Markers of viral replication in the CNS in living subjects must come from the analysis of virus in the CSF, or be inferred from indirect markers from non-invasive imaging techniques or from cell or inflammatory markers in the CSF or blood.Virus can be found in the CSF as the result of three distinct mechanisms. InfectedT cells traffic into the CNS and release virus that by sequence looks just like the virus in the blood, and this virus can be present at a low level or, with pleocytosis, at a high level. Alternatively, some of this virus can be transiently clonally amplified by a poorly understood mechanism to give elevated viral loads but of surprisingly homogeneous viral populations. Finally, virus can take root within the CNS for sustained replication but in an environment that is poor in CD4+T cells.This causes the virus to adapt to macrophage-like cells with their low levels of the surface CD4 protein needed for entry.The unique genetic lineage of virus replicating independently in the CNS and the phenotypic adaptation to this CD4 low environment provide clear evidence of a distinct viral population and site of ongoing viral replication.Therapy suppresses viral load in the blood and the CSF. However, antiviral drug levels are reduced in the CNS, a situation that would result in viral replication and the selection of resistance if it took place in the blood. In about 5-10% of subjects on therapy there is detectable virus in the CSF either in the absence of or at levels higher than virus in the blood.This CSF escape virus takes on several forms, including a complex viral population at least partially adapted to a CD4 low environment.We do not know how well the CSF records virologic events that may be ongoing deep within the brain parenchyma. Such replication when detected in the CSF represents evidence of an active viral reservoir on therapy, while the potential for a unique latent reservoir within the CNS is of considerable interest but challenging to study. 63 Highly Active Antiretroviral Treatment, despite its striking systemic efficacy, might be incompletely effective in some organs and tissues thus favouring residual HIV replication and damage. The central nervous system (CNS) is a key organ in this setting and several data have reported a worrisome prevalence of neurocognitive deficits in effectively treated HIV-positive patients. In this talk data on the potential mechanisms underlying CNS disease in HIV-positive patients including early treatment, residual HIV replication, Therapeutics of the CNS With HIV Infection Andrea Calcagno , Univ of Torino, Torino, Italy

Oral Abstracts

25

CROI 2016