HIV-1 Vpr redirects host ubiquitination pathway. Sakshi Arora1#, Sachin Verma2 and Akhil C ...

36 Pages·2014·0.92 MB·English

JVI Accepts, published online ahead of print on 4 June 2014
J. Virol. doi:10.1128/JVI.00619-14
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1 HIV-1 Vpr redirects host ubiquitination pathway.
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3 Sakshi Arora1#, Sachin Verma2 and Akhil C. Banerjea1#
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5 1Laboratory of Virology, National Institute of Immunology, New Delhi-110067, India. o
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6 2Salk institute for Biological studies, La jolla, CA, USA. lo
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8 Sakshi Arora- [email protected]; Sachin Verma - [email protected]. m

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10 Running Title: Vpr reduces whole cell ubiquitination in host cells. jvi.
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12 #Address correspondence to Akhil C. Banerjea; [email protected] & [email protected] and Sakshi /

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13 Arora; [email protected] N
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23 Abstract
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25 HIV-1 modulates key host cellular pathway for successful replication and pathogenesis through
26 viral proteins. Evaluating the hijacking of the host ubiquitination pathway by HIV-1 at whole cell
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27 level, we now show major perturbations in the ubiquitinated pool of the host proteins post HIV- o
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28 1 infection. Our over-expression and infection based studies in T-cells with wild type and lo
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29 mutant HIV-1 proviral constructs showed that Vpr is necessary and sufficient for reducing whole d

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30 cell ubiquitination. Mutagenic analysis revealed that the leucine rich three helical regions of Vpr m

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31 is critical for this novel function of Vpr which was independent of its other known cellular tp
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32 functions. We also validated that this effect of Vpr was conserved among different subtypes, (B jvi.
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33 and C) and circulating recombinants from Northern India. Finally, we establish that this m
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34 phenomenon is involved in HIV-1 mediated diversion of host ubiquitination machinery /

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35 specifically towards degradation of various restriction factors during viral pathogenesis. N
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37 HIV-1 is known to rely heavily on modulation of host ubiquitin pathway particularly for e
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38 counteraction of antiretroviral restriction factors i.e., APOBEC3G, UNG2, BST-2 etc., viral ,

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39 assembly and release. Reports till date have focused on molecular hijacking of ubiquitin 8

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40 machinery by HIV-1 at the level of E3 ligases. Interaction of a viral protein with an E3 alters its
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41 specificity to bring about selective protein ubiquitination. However, in case of infection, s
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42 multiple viral proteins can interact with this multi-enzyme pathway at various levels making it
43 much more complicated. Here, we have addressed manipulation of ubiquitination at the whole
44 cell level post HIV-1 infection. Our results show that HIV-1 Vpr is necessary and sufficient to
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45 bring about redirection of host ubiquitin pathway towards HIV-1 specific outcomes. We, also
46 show that the leucines rich three helical region of Vpr is critical for this effect and this ability of
47 Vpr is conserved across circulating recombinants. Our work, first of its kind, provides novel
48 insight into regulation of ubiquitin system at whole cell level by HIV-1.
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67 Introduction
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69 Human Immunodeficiency Virus type 1 (HIV-1), a primate lentivirus, primarily infects T-cells,
70 macrophages and probably dendritic cells. This narrow tropism is determined by the cell
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71 surface receptors (CD4 and co-receptor CXCR4/CCR5) required for HIV-1 to attach and gain o
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72 entry (1). HIV-1 infection is characterized by a gradual deterioration in immune function lo
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73 because of severe depletion of CD4-T lymphocytes that ultimately causes AIDS in humans (2). d
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74 A human cell harbors a number of host encoded antiretroviral restriction factors to ensure m

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75 protection from invading retroviruses. HIV-1 on the other hand being a highly evolved tp
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76 retrovirus has mechanisms to evade these restrictive host responses. A detailed understanding jvi.
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77 how the virus establishes successful infection despite the presence of numerous antiretroviral m
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78 factors is essential for identifying and developing effective therapeutics and vaccines. /

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80 HIV-1 genome is unique compared to other retroviruses because of the presence of highly e
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81 evolved accessory genes (Vpr, Vif, Nef and Vpu) (3). Most of these small open reading frame e
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82 (ORF) encoded proteins are involved in manipulating cellular physiology for immune evasion, ,

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83 replication and transmission (4). In addition these accessory proteins confer HIV-1 exceptional 8

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84 ability to overcome numerous cellular antiretroviral restriction factors (ARVs) (4, 5) present
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85 throughout the viral life cycle. Targeted degradation of specific restriction factors during s
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86 infection is achieved by diversion of cellular ubiquitin proteosomal pathtway (UPP) by viral
87 accessory proteins. UPP involves a multi enzyme cascade with three distinct enzymes namely
88 E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme) and E3 (ubiquitin ligase).
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89 The substrate specificity is mediated at the level of E3 which are further classified into three
90 groups: RING, HECT and F-box containing ligases (6). The sequential attachment of Ub to
91 various cellular proteins is also regulated by deubiquitinases (Cysteine proteases), that remove
92 Ub from proteins (7). Attachment of ubiquitin is a reversible event that is induced by various
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93 stimuli which not only affects protein stability but also regulates functional interactions thus o
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94 controlling various cellular processes like localization, proliferation and immune responses (8). lo
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95 Ability of ubiquitinated proteins to play myriad of functions depends on number of ubiquitin d

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96 molecules attached and the type of linkage. Mono-ubiquitination regulates vesicular transport, m

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97 DNA repair and virus budding (9). Polyubiquitination at K48 mediates protein degradation, cell tp
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98 cycle arrest, and the same at K63 regulates activation of protein kinases and DNA repair (9, 10). jvi.
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99 All these host cellular processes are of prime interest with respect to viral pathogenesis hence, m
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100 viruses have developed mechanisms to exploit the UPP to create cellular state favorable to /

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101 their replication and pathogenesis (8, 11). N
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103 Viruses may either encode ubiquitin, E3 ligases or deubiquitinases in their genome. In addition, e
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104 often viral proteins act as adaptors altering the specificity of E3 ligases to bring about specific ,

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105 protein ubiquitination thereby hijacking the cellular Ub ligase complex (11). During HIV-1 8

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106 infection, degradation of antiretroviral factor APOBEC3G requires association of Vif protein with
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107 cullin-5 ElonginB-ElonginC complex (12-17). Vpr-mediated G2 arrest involves the DDB1-CUL4A s
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108 (VPRBP) E3 ubiquitin ligase (18-20) that is essential for viral replication. Furthermore,
109 degradation of interferon induced BST-2/Tetherin and CD4 by Vpu protein depends on the
110 ability of Vpu protein to bind (cid:628)-TrCP subunit of SCF (Skp1-Cullin-F-box)-(cid:628)-TrCP ubiquitin ligase
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111 complex (21-23). In addition, HIV-1 Nef is multiply ubiquitinated which is critical for CD4 down-
112 regulation (24) and Gag is ubiquitinated that is essential for virus budding (25). The diversion of
113 E3 substrate specificity by viral proteins in certain cases can be so drastic that the natural
114 function of Ub-ligase complex is inhibited or compromised. Previous reports support this notion
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115 and show that interaction of Vpu with SCF-(cid:628)-TrCP E3 complex results in accumulation of many o
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116 of its natural substrates ((cid:628) catenin, ATF4, IkB and p53) contributing to viral pathogenesis (26- lo
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117 29). UPP is also critical for transactivation (30), NF-kB pathway (31) as well as assembly and d

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118 release of virions from infected cell (32). Hence, exploitation of UPP via extensive interplay m

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119 between multiple viral proteins and different cellular Ub-ligase complexes seems to play a tp
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120 major role in driving HIV-1 pathogenesis. The broad cellular consequences resulting from these jvi.
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121 changes within infected cells however remain unidentified. m
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123 In the present report, we show that the whole cell ubiquitination is reduced within HIV-1 N
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124 infected cells. The ubiquitination of known ARVs, however, was found to be protected from this e
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125 inhibition. Interestingly, Vpr contributes to this major perturbation in pool of ubiquitinated e
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126 proteins during infection as well as in over-expression assays. The structural integrity of the ,

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127 three helical domains of Vpr was found to be critically important for this newly identified 8

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128 function conserved across various subtypes and primary isolates of virus. In agreement with
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129 previously demonstrated ubiquitination of ARV factors by numerous viral proteins, this s
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130 inhibitory role of Vpr on host-specific cellular ubiquitination possibly suggest redirection of
131 host ubiquitin pathway towards specific outcomes important for HIV-1 infection i.e.,
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132 degradation of antiviral factors. This study is first of its kind where effect of a viral infection on
133 whole cell ubiquitination has been pursued from a host perspective.
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149 Materials and Methods
150 Plasmid constructs and proviral DNAs
151 Vpr, Tat, Rev, Nef, Vpu and Vif from subtype B (pNL4-3) and Vpr from subtype C (Indian Isolate
152 93IN905) HIV-1 (obtained from National Institute of Allergy and Infectious Diseases [NIAID], D
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153 National Institutes of Health [NIH]) were amplified by PCR and cloned in the mammalian lo
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154 expression vector pCMV-Myc (Clontech) to generate Myc-Vpr B, Myc-Tat B, Myc-Nef B, Myc- e
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155 Vpu B, Myc-Vif B and Myc-Vpr C constructs. The Myc-VprB L22A, L64A, L64P, L67A, R62P, R80A, ro
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156 (cid:564)17-33, (cid:564)38-48 and (cid:564)53-77 mutants were generated by site directed mutagenesis using Kappa t
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157 Hi-Fi DNA Polymerase (Kappa Biosystems). HA-APOBEC3G was cloned using cDNA from Tzmbl /jv
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158 cells. The pNL4-3 HIV-1 clone as well as mutant pNL4-3 (cid:564)vpr (having deletion in Vpr initiation s
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159 codon) variants were kind gifts from K. Strebel (NIH) (26). pNL4-3 (cid:564)vpr(cid:564)vif ((cid:1104)VV) was a kind gift r
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160 from Kathleen Boris-Lawrie (33) UNG2-HA was a kind gift from Dr. Serge Benichou (34). n

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161 Cell culture, transfections, and immunoblot analysis e
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162 HEK 293T (Human Embryonic Kidney 293T) and TZM-bl cells were maintained in DMEM 1
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163 (HiMedia) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U(cid:1064)mL penicillin and 2
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164 100 (cid:645)g(cid:1064)mL streptomycin (Invitrogen) at 37°C with 5% CO . Jurkat E6.1 T cell (leukemic T cell
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165 lymphoblast) were maintained in RPMI (HiMedia) media supplemented with glutamine, 10% u
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166 FBS, 100 U(cid:1064)mL penicillin and 100 (cid:645)g(cid:1064)mL streptomycin (Invitrogen) at 37°C with 5% CO . Plasmid t
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167 transfections were performed using Lipofectamine 2000 (Invitrogen) as per the manufacturer’s
168 protocol. Viral stocks of pNL4-3, pNL4-3(cid:564)vpr, pNL4-3(cid:564)vif and pNL4-3 (cid:564)vpr(cid:564)vif ((cid:1104)VV) were
169 prepared by co-transfecting different proviral DNAs and plasmid-encoding Vesicular Stomatitis
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170 Virus Glycoprotein (VSV-G) into HEK 293T cells, followed by collection of viral particles from
171 culture supernatant at 48 and 72 hours. The levels of different cellular proteins were compared
172 by immunoblot analysis. Cells were lysed in RIPA Lysis buffer (Cell Signalling Technology) and
173 protein estimation was done using BCA Protein Estimation Kit (Pierce Biotechnology, Inc.). The
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174 primary antibodies used were anti-Myc, anti-HA (Clontech), anti-Ub, anti-GAPDH (Cell Signaling o
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175 technology), anti-His (Sigma-Aldrich), anti-p24 (Cat No. 6457, NIH) and anti-tubulin (Santa Cruz lo
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176 Biotechnology, Inc). The secondary antibodies used were anti-rabbit/mouse-HRP conjugated d

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177 (Jackson Immuno Research) and anti-mouse-FITC conjugated (BD Biosciences). The proteins of m

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178 interest were detected with EZ western horse radish peroxidase substrate (Biological Industries, tp
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179 Israel). GAPDH was used as a loading control. jvi.
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180 In vivo ubiquitination assay .o
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181 For detection of whole cell ubiquitination, HEK 293T cells were grown in 35-mm dishes and n

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182 transfected with 1μg of 6X His-ubiquitin expression plasmid (35) along with equal amounts of v
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183 various indicated plasmids. After 36 hours of transfection, 25 μM of MG132 (Sigma-Aldrich) was b
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184 added, and cells were further incubated for 8 hours. Thereafter, cells were collected in PBS and 7
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185 were resuspended in 1 mL of lysis buffer (6M guanidinium-HCl, 0.1M Na HPO /NaH PO , 0.01M 0
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186 Tris, pH 8.0, 10mM imidazole, and 10mM (cid:628)-Mercaptoethanol), sonicated, and centrifuged. To y

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187 equal amount of cell lysate Ni-NTA beads (50 μL) were added and the mixture was incubated at e
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188 room temperature for 4 hours while rotating. Subsequently, the beads were washed for 5
189 minutes at room temperature with 750 μL of each of the following buffers: lysis buffer; buffer A
190 (1.5M guanidium-HCl, 0.025M Na HPO /NaH PO4, 0.01M Tris pH 8.0, 10mM (cid:628)-
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191 Mercaptoethanol; buffer B (0.025 M Tris pH 6.8, 20mM Imidazole 0.2% Triton X-100).
192 Ubiquitinated proteins were eluted by incubating the beads in 75 μL of buffer containing
193 200mM imidazole, 5% SDS, 0.15M Tris, pH 6.7, 30% glycerol, 0.72M (cid:628)-Mercaptoethanol for 20
194 minutes at room temperature. The elutes were mixed in a 1:1 ratio with 2X Laemmli buffer and
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195 resolved by SDS-PAGE followed by immunoblotting with indicated antibodies. o
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196 Infection by HIV-1 pNL4-3 or HIV-1 mutants d
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197 Jurkat E6.1 T, HEK 293T and TZM-bl cells were infected with pNL4-3/pNL4-3(cid:1104)vpr/pNL4- ro
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198 3(cid:1104)vif/pNL4-3(cid:1104)vifvpr ((cid:1104)VV) viral stocks. Infection was accomplished by incubating cells for 4 t
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199 hours with equal amounts of infectious virus as assessed by (cid:628)-galactosidase staining using HIV-1 /jv
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200 indicator Tzmbl cells (36). The infected cells were harvested 48 hours after infection and s
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201 divided into two halves. One half was subjected to immunoblotting with indicated antibodies. r
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202 Other half of cells was evaluated for the extent of HIV-1 infection by intracellular p24 staining n

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203 with primary p24 mouse antibody (Cat No. 6457, NIH) followed by secondary anti-mouse v
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204 antibody (FITC conjugated). The intracellular p24 levels of infected cells were assessed by flow b
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205 cytometer. 7
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206 Cell cycle staining 8

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207 HEK 293T cells were collected 48 hours post transfection. They were fixed in 70% alcohol and u
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208 kept at 4(cid:1014) overnight. There were then washed twice with 1X PBS and stained with 10μg/ml of t
209 propidium iodide. The cells were then analyzed on BD FACSVerse.
210 Luciferase Assay
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