Human cytomegalovirus UL97 kinase and nonkinase functions mediate viral cytoplasmic secondary envelopment.
Previous studies have revealed critical roles for the human cytomegalovirus (HCMV) UL97 kinase in viral nuclear maturation events. We have shown recently that UL97 affects the morphology of the viral cytoplasmic assembly compartment (AC) (M. Azzeh, A. Honigman, A. Taraboulos, A. Rouvinski, and D. G. Wolf, Virology 354:69-79, 2006). Here, we employed a comprehensive ultrastructural analysis to dissect the impact of UL97 on cytoplasmic steps of HCMV assembly. Using UL97 deletion (ΔUL97) and kinase-null (K355M) mutants, as well as the UL97 kinase inhibitor NGIC-I, we demonstrated that the loss of UL97 kinase activity resulted in a unique combination of cytoplasmic features: (i) the formation of pp65-rich aberrant cytoplasmic tegument aggregates, (ii) distorted intracytoplasmic membranes, which replaced the normal architecture of the AC, and (iv) a paucity of cytoplasmic tegumented capsids and dense bodies (DBs). We further showed that these abnormal assembly intermediates did not result from impaired nuclear capsid maturation and egress per se by using 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl) benzimidizole (BDCRB) to induce the artificial inhibition of nuclear maturation and the nucleocytoplasmic translocation of capsids. The specific abrogation of UL97 kinase activity under low-multiplicity-of-infection conditions resulted in the improved release of extracellular virus compared to that of ΔUL97, despite similar rates of viral DNA accumulation and similar effects on nuclear capsid maturation and egress. The only ultrastructural correlate of the growth difference was a higher number of cytoplasmic DBs, tegumented capsids, and clustered viral particles observed upon the specific abrogation of UL97 kinase activity compared to that of ΔUL97. These combined findings reveal a novel role for UL97 in HCMV cytoplasmic secondary envelopment steps, with a further distinction of kinase-mediated function in the formation of the virus-induced AC and a nonkinase function enhancing the efficacy of viral tegumentation and release.
Hadassah Hebrew University Medical Center, Jerusalem, Israel.
Miri D
MD
Goldberg
Alik
A
Honigman
Jacob
J
Weinstein
Sunwen
S
Chou
Albert
A
Taraboulos
Alexander
A
Rouvinski
Vera
V
Shinder
Dana G
DG
Wolf
D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000201:enzymology* / Q000502:physiology* / Q000648:ultrastructure; D005347:Fibroblasts / Q000821:virology; D017353:Gene Deletion; D006801:Humans; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly
D050505:Mutant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase
Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6701-5
8393574
Blood. 2003 Jan 15;101(2):463-5
12393485
Science. 2008 May 9;320(5877):797-9
18467589
J Virol. 2000 May;74(9):4004-16
10756012
Eur J Cell Biol. 1993 Feb;60(1):163-78
8385018
J Virol. 1999 Sep;73(9):7271-7
10438815
J Gen Virol. 2010 Oct;91(Pt 10):2531-41
20592110
J Virol. 2003 Oct;77(19):10594-605
12970444
PLoS Pathog. 2009 Jan;5(1):e1000275
19165338
Virology. 2003 Jun 20;311(1):60-71
12832203
J Virol. 2003 Jan;77(2):905-14
12502806
J Virol. 2002 Feb;76(4):1537-47
11799148
J Clin Microbiol. 2004 Mar;42(3):1142-8
15004066
J Virol. 2001 Aug;75(15):6865-73
11435566
J Virol. 1994 Mar;68(3):1350-9
8107200
Virol J. 2009 Jan 21;6:9
19159461
Virology. 2006 Oct 10;354(1):69-79
16872656
J Virol. 2007 Oct;81(19):10659-68
17634236
Virology. 1997 Apr 28;231(1):72-80
9143304
J Virol. 2003 May;77(9):5339-51
12692236
J Biol Chem. 2002 Aug 16;277(33):29593-9
12048183
J Virol. 2010 Apr;84(7):3162-77
20089649
J Virol. 1999 Oct;73(10):8320-9
10482582
J Med Chem. 2006 Nov 30;49(24):7044-53
17125257
J Gen Virol. 2009 Mar;90(Pt 3):579-590
19218202
J Virol. 1999 Jul;73(7):5663-70
10364316
J Virol. 2004 Mar;78(6):3024-34
14990721
Traffic. 2008 Sep;9(9):1458-70
18564370
Antiviral Res. 2006 Sep;71(2-3):154-63
16765457
Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1895-900
11172047
J Virol. 2008 Dec;82(23):11913-26
18815303
Traffic. 2002 Mar;3(3):218-32
11886592
Mol Biol Cell. 2008 Sep;19(9):3871-84
18614794
Antimicrob Agents Chemother. 2009 Jan;53(1):81-5
18981262
J Virol. 2003 Jul;77(14):7720-7
12829811
Science. 2002 Aug 2;297(5582):854-7
12161659
Virology. 2004 Jun 20;324(1):184-93
15183065
J Virol. 2008 May;82(10):5054-67
18321963
J Biol Chem. 2005 Sep 30;280(39):33357-67
15975922
J Gen Virol. 2001 Jun;82(Pt 6):1439-1450
11369889
J Gen Virol. 2007 Oct;88(Pt 10):2642-2650
17872514
J Gen Virol. 2009 Mar;90(Pt 3):567-578
19218201
J Virol. 2000 Jan;74(2):975-86
10623760
J Virol. 2007 Nov;81(21):11861-9
17715239
J Virol. 2007 Jun;81(12):6536-47
17392372
J Virol. 2006 Feb;80(3):1476-86
16415024
PLoS Pathog. 2007 Jan;3(1):e6
17206862
J Virol. 2005 Dec;79(24):15494-502
16306620
J Virol. 2007 Oct;81(19):10316-28
17626081
J Virol. 1999 Jul;73(7):5364-72
10364283
Curr Top Microbiol Immunol. 2010;342:99-111
20186612
J Virol. 2003 Mar;77(5):3191-203
12584343
J Virol. 2006 Aug;80(16):8199-210
16873276
Clin Infect Dis. 2008 May 1;46(9):1455-7
18419454
J Virol. 2008 Oct;82(19):9433-44
18653449
J Virol. 1996 Sep;70(9):6340-6
8709262
Nucleic Acids Res. 2005 Feb 24;33(4):e36
15731329
Traffic. 2009 Oct;10(10):1439-57
19761540
J Virol. 2008 Mar;82(6):2802-12
18184717
J Virol. 1998 Jan;72(1):717-25
9420278
J Virol. 2004 Oct;78(20):10960-6
15452216
J Virol. 2004 Jan;78(1):539-43
14671136
Antiviral Res. 2002 Oct;56(1):61-72
12323400
Proc Natl Acad Sci U S A. 1998 Sep 29;95(20):11969-74
9751774
J Virol. 2000 Apr;74(7):3093-104
10708424
J Cell Sci. 2005 May 1;118(Pt 9):1991-2003
15840653
J Virol. 2009 Mar;83(6):2480-90
19116255
Biochim Biophys Acta. 2004 Mar 11;1697(1-2):169-80
15023359
Rev Med Virol. 2009 Jul;19(4):215-29
19434630
J Virol. 2007 May;81(10):5212-24
17229708
J Virol. 2009 Oct;83(20):10797-807
19640981
Nature. 1992 Jul 9;358(6382):162-4
1319560
Antimicrob Agents Chemother. 2002 Aug;46(8):2365-72
12121906
J Virol. 2004 Feb;78(3):1488-502
14722304
J Virol. 1999 May;73(5):4456-60
10196346
J Virol. 2010 Jul;84(14):7005-17
20484513
Virology. 2009 Sep 15;392(1):137-47
19640559
Curr Top Microbiol Immunol. 2010;342:79-98
20186610
Antimicrob Agents Chemother. 2005 Jul;49(7):2710-5
15980340
Nature. 1992 Jul 9;358(6382):160-2
1319559