The Role of Host Factors in HIV-1 Entry and Assembly Open Access

Abstract

HIV-1 is a global health threat with no vaccine available. Current treatment regiments are generally effective, but life-long administration of antiretroviral drugs has side effects and is quite costly. Uncovering the molecular mechanisms underlying the HIV-1 life cycle is important for developing effective drugs. HIV-1 Env is the only viral surface protein and it functions by initiating fusion to the host cell membrane, thereby initiating infection. Env is a major target for neutralizing antibodies. However, due to a dense glycan shield covering the Env and complex conformational changes during the fusion process, access to Env neutralization epitopes is limited. We therefore examined how Env gets incorporated into virions and how it induces viral fusion with the host membrane. To incorporate into progeny virions, Env must be transported to the virus assembly sites on the plasma membrane and interact with the assembling structural Gag protein. Here we elucidated the role of Rab11 and FIP1C in Env trafficking during HIV-1 assembly (Chapter 2). We found that the C-terminal fragment of FIP1C, when expressed in cells, can sequester Env inside the endosomal recycling compartment and thereby block the Env incorporation into virions. This dominant negative effect is likely proceeded by endocytosis of Env from the plasma membrane and is dependent on tyrosine motifs in the Env cytoplasmic tail. We also examined the mechanism of restriction of HIV-1 fusion by the new restriction factor SERINC5 (Chapter 3). SERINC5 incorporates into virions in the absence of Nef and potently inhibits fusion of sensitive, but not resistant HIV-1 variants. Our results revealed that both sensitive and resistant Env glycoproteins are functionally inactivated over time and that the inactivation rate is increased upon SERINC5 incorporation into virions. Mutations that stabilize or destabilize specific regions on Env did not reveal a specific Env conformation that could be targeted by SERINC5. However, a CD4 mimetic compound accelerated SERINC5-mediated Env inactivation, while a conformational blocker that stabilizes a native Env structure delayed the functional inactivation of Env in the presence of SERINC5. Collectively, our studies provided new insights into the molecular mechanisms of HIV-1 interaction with the host proviral and restriction factors during virus assembly and entry into target cells.

Table of Contents

Chapter I: Introduction ....................................................................................................................... 1

   Figures .............................................................................................................................................. 19

     Fig 1. HIV-1 replication cycle…………………………………................................................................... 19

     Fig 2. HIV-1 Env incorporation model................................................................................................. 20

     Fig 3. HIV-1 Env CT …………………………................................................................................................. 21

   References ....................................................................................................................................... 22

Chapter II: Dominant negative effect of C-terminal domain of FIP1C on HIV-1 Env ... 49

   Abstract ........................................................................................................................................... 50

   Introduction ................................................................................................................................... 51

   Results .............................................................................................................................................. 64

   Discussion ....................................................................................................................................... 62

   Materials and Methods ............................................................................................................... 72

   Figures ....................................................................................................................................................... 77

     Fig. 1. Truncated FIP1C inhibits Env incorporation and particle infectivity...................... 77

     Fig. 2. FIP1C560–649 sequesters Env in a perinuclear compartment in a CT-dependent 

     manner.............................................................................................................................................................. 79

     Fig. 3. Cell surface levels of CT144 Env and effect of FIP1C560–649 versus FIP2452–512...... 81

     Fig. 4. Trapping of Env by GFP-FIP1C560–649 is dependent on specific trafficking motifs 

     in the CT………………………………………………………………………………………………....................... 83

     Fig. 5. Trapping of Env by GFP-FIP1C560–649 is dependent on specific trafficking  motifs 

     in the CT. ………………………………………………………............................................................................ 85

     Fig. 6. Dominant-negative FIP1C560–649 fails to trap SIV Env in the ERC. ............................. 87

     Fig 7. Dose titration of FIP1C560–649 and effects on SIVmac239 Env incorporation ........ 89

     Fig 8. Particle infectivity correlates with replicative capacity ................................................ 91

     Fig 9. Endosomal markers present in FIP1C560–649-positive ERC. .......................................... 93

     Fig 10. Endocytosis and trapping of artificial Env in ERC ......................................................... 95

     Fig 11. Model for Env trafficking and retention in ERC by truncated FIP1C…………....... 97

     S1 Fig. Endocytosis and trapping of natural Env in ERC…......................................................... 99

     S2 Fig. Rab14 binding site in FIP1C560–649 is responsible for Env trapping.......................101

     S3 Fig. FIP1C560–649 can isolate AD8-Env in the ERC in human macrophage................... 103   

   References .................................................................................................................................... 105

Chapter III: The mechanism of HIV-1 restriction by SERINC5 ……………………………..… 117

   Abstract ......................................................................................................................................... 118

   Introduction ................................................................................................................................ 119

   Results ........................................................................................................................................... 130

   Discussion .................................................................................................................................... 140

   Materials and Methods ............................................................................................................ 145

   Figures ........................................................................................................................................... 150

     Fig. 1 SERINC5 accelerates spontaneous inactivation of sensitive and, to a lesser extent,  

           of resistant Env............................................................................................................................... 150

     Table 1. Spontaneous inactivation of sensitive Envs is more profoundly accelerated by   

          SERINC5 than resistant Envs..................................................................................................... 151

     Fig. 2 The rate of functional Env inactivation by SERINC5 at 37°C weakly correlates 

          with sensitivity to restriction.................................................................................................... 152

     Fig. 3. Mutations that destabilize or stabilize the native HIV-1 Env structure do not 

          consistently alter the anti-viral activity of SERINC5.........................................................155

     Fig. 4 The effect of Env-stabilizing mutations the SERINC5 activity................................... 156

     Fig. 5 Increased density of HIV-1 Env reduces sensitivity to SERINC5 ........................... 157

     Fig. 6 Constraining native/ground state of Env by 484 does not significantly change 

          JRFL Env sensitivity to SERINC5. ............................................................................................ 159

     Table 2. Relationship between Env stability, SERINC5-sensitivity and sensitivity to 484 

          inhibition. .......................................................................................................................................... 160

     Fig.7 Effect of Env-stabilizing the destabilizing mutations on SERINC5- and 484-

         sensitivity............................................................................................................................................. 161

     Fig. 8 CD4 mimetic augments SERINC5-mediated acceleration of infectivity decay ... 162

     Fig. 9 Conformational blocker BMS-378806 decelerates SERINC5-mediated decay of 

         JRFL infectivity.................................................................................................................................... 164

   References .................................................................................................................................... 165

Chapter IV: General Discussion…………………………..………...................................................... 184

   References .................................................................................................................................... 193

Appendix ............................................................................................................................................ 196

     Fig 1. Rab11-Family Interacting Proteins (FIPs)………………………………………………….. 196

     Fig. 2. Endogenous expression of endocytic pathway markers........................................... 197    

     Fig. 3. Cellular markers with RBD-FIP2 …………………............................................................... 299

     Fig. 4. Cellular markers with WT-FIP1C …………………............................................................... 200

     Fig. 5. Early expression of FIP1C549-650 ………………….................................................................. 201

     Fig. 6. Fig. 6 RBD-FIP1C and HIV2 Envs ……………….................................................................. 202

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Cell Biology, Microbiology Biology, Virology
Keyword	envelope HIV-1 restriction SERINC5 endosomal recycling compartment stability conformation virus entry virus assembly Rab11-FIP1C
Committee Chair / Thesis Advisor	Gregory Melikian, Emory University
Committee Members	Anice Lowen, Emory University Daniel Kalman, Emory University Baek Kim, Emory University Eric Hunter, Emory University

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