HIV Cellular Pathogenesis II Benhur Lee, M.D.

HIV Accessory Genes: Tat Rev Vif Vpr Vpu Nef Essential in vitro and in vivo Essential in certain cell types (Permissive vs Non-permissive cells) Non-essential in vitro, but leads to attenuated phenotype in vivo

Nef Major determinant of pathogenicity in vivo nef-deleted SIV is severely attenuated in the rhesus macaque model infection of macaques with recombinant SIV carrying a premature STOP codon (point mutation) results in rapid revertants with the nef ORF Patients infected with nef-defective HIV have a dramatically decreased rate of disease progression (>15 years) nef-deleted HIV do not deplete thymocytes as much, or replicate to as high titers, as wild-type HIV in the SCID-hu mice model

Pleiotropic Functions of Nef N-myristoylation required for Nef activity--implies that membrane localization of nef is critical for its activity Consensus N-myristoylation Signal: MGxxx(S/T)(K/R)(K/R) MGxxx(S)(K)(K/R) HIV sequence Conservation in Nef protein: 100% 99% 100% ~50%

Pleiotropic Functions of Nef Down-regulates cell surface levels of CD4 Down-regulates surface levels of major histocompatibility class I molecules Mediates cellular signaling and activation Enhances viral infectivity

I. Down-modulation of surface CD4 Down-modulation of surface CD4 via internalization followed by degradation via endosomal/lysosomal pathway Advantages: Prevents disadvantageous super-infection of host cell Enhance viral progeny release (by preventing Env-mediated sequestration of CD4 in secretory pathway) Evidence: Nef expression increases number of CD4 containing clathrin-coated pits Nef-induced CD4 down-modulation blocked by inhibitors of clathrin-coated pit-mediated endocytosis (e.g. ikaguramycin) Inhibition of lysosomal acidification (e.g. via chloroqine treatment) blocks Nef-induced CD4 degradation Expression of nef alone in T-cell lines can lead to CD4 downregulation (as determined by FACS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CD4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nef-GFP

I. Down-modulation of surface CD4 Mechanism(s)? Direct interaction with CD4 has not been biochemically demonstrable, but NMR analysis suggest a direct interaction with Kd ~0.87 mM; yeast two-hybrid assays also suggest an interaction Acts as a connector to the host-cell endocytic machinery Co-localizes with AP-2 on inner plasma membrane Conserved dileucine based sorting motif (E/DxxxL) in Nef is important for both CD4-down-modulation and AP-2 co-localization Interacts with NBP-1 (identified through a yeast two-hybrid screen). NBP-1 is part of the vacuolar membrane ATPase complex in clathrin-coated pits (H subunit of vacuolar ATPase--VH1) C-terminal diacidic motif (DD) in Nef is important for NBP-1 interaction, and, at least in SIV Nef, the dileucine motif is also important for NBP-1 interactions ?? May bind to b-Cop, a coatamer protein which targets proteins to lysosomes NBP-1

II.Down-modulation of MHC Class I Advantages: Immune evasion; MHC Class I presents antigens to cytotoxic T- lymphocytes; alerts innate and adaptive immune system to virally infected cells Evidence: Nef expression reduces susceptibility of HIV-infected cells to CTL mediated lysis in vitro selectively down-regulates only HLA-A and HLA-B, which presents antigens to CTLs; does NOT down-regulate HLA-C and HLA-E, which inhibit NK-cell mediated cell lysis Thus, efficiency of CTL-mediated lysis (adaptive immunity) is reduced without increasing increasing susceptibility to NK cell lysis CTL HIV MHC Class I HIV antigen 51Cr

100% HIV Dnef HIV wt % Lysis E (Effector Cell) CTL 0% 1:2 1:5 1:10 1:20 MHC Class I HIV antigen E:T ratio T (Target Cell)

III. Mediates Cellular Activation and Signaling Nef expression upregulates a transcriptional program that activates the HIV LTR (microarray analysis)

III. Mediates Cellular Activation and Signaling Nef expression upregulates a transcriptional program that activates the HIV LTR (microarray analysis) Nef can induce secretion of paracrine factors that enhance viral replication; macrophage supernatants from cells transduced with nef-expressing adenoviral vector can facilitate HIV replication in resting lymphoid cultures Adv-nef supnt p24 (ng/ml) Adv-GFP supnt 3 6 9 (days)

III. Mediates Cellular Activation and Signaling Nef expression upregulates a transcriptional program that activates the HIV LTR (microarray analysis) Nef can induce secretion of paracrine factors that enhance viral replication; macrophage supernatants from cells transduced with nef-expressing adenoviral vector can facilitate HIV replication in resting lymphoid cultures Nef interacts with Pak2 (p21 activated kinase 2) and Nef/Pak2 complex may regulate many of Nef’s effect on gene transcription

IV. Infectivity Enhancement Magnitude of infectivity enhancement is allele dependent Nef mediated enhancement can be provided in trans reporter gene (e.g. GFP or luciferase) expression under control of the LTR promoter can be enhanced when nef expression vector is co-transfected Mechanisms: Increased RT activity; increased proviral DNA synthesis Increased cytoplasmic delivery of viral particles

Vif: Viral infectivity factor, required for robust replication only in certain cells C8166, 293T, HeLa Hut78, H9, 1º PBLs Permissive Non-permissive HIV-1 ( +++ replication +++ replication HIV-1 (Dvif) +++ replication no replication Two hypotheses: Permissive cells express an activity (factor) that can compensate for vif. Non-permissive cells have an inhibitory activity on viral replication, which is overcomed by vif. See Simon et. al., Nature Med. 4: 1397

Heterokaryon Non-permissive Permissive wt Dvif wt Dvif wt Dvif ++ - ++ Which phenotype will dominate? Non-permissive Permissive wt Dvif wt Dvif ++ - ++ Non-permissive: inhibitory cellular factor overcomed by vif Permissive: compensatory factor similar to vif Infectivity

Denv vs Denv/Dvif Non-Permissive Permissive Two hypotheses: Permissive (ensures that all viruses are produced from Heterokaryons) Two hypotheses: Permissive cells express an activity (factor) that can compensate for vif. Non-permissive cells have an inhibitory activity on viral replication, which is overcomed by vif. Permissive

Heterokaryon Non-permissive Permissive wt Dvif wt Dvif wt Dvif ++ - ++ Non-permissive: inhibitory cellular factor overcomed by vif Infectivity

CEM-SS (permissive cells) renders it non-permissive Expression of CEM15 in CEM-SS (permissive cells) renders it non-permissive After subtractive-PCR for differentially expressed genes

CEM15=APOBEC3G (Apolipoprotein B mRNA Editing enzyme, Catalytic polypeptide-like 3G) APOBEC3G, cytosine deaminase APOBEC1, cytidine deaminase involved in mRNA editing AID (Activation-induced cytidine deaminase), involved in somatic hypermutation, required for immunoglobulin gene diversification Incorporated into virions Deaminates cytosines to uracils during first strand cDNA synthesis in target cells Moderate sequence specificity (Py-C-C) Degradation: uracil in DNA recognized by host uracil DNA glycosidase, which removes uracil from DNA-->cleavage and degradation Hypermutation: massive C-->U conversion on minus strand leads to massive G-->A hypermutation of the plus strand; resultant provirus is so heavily mutated, no functional viral proteins are encoded

How does vif overcome APOBEC3G’s antiviral function? Reduces the level of APOBEC3G in producer cell Expression of vif reduces APOBEC3G levels Vif promotes degradation of APOBEC3G via Ubiquitin-proteasome pathway Vif impairs efficient translation of APOBEC3G mRNA