1
1
Contributions of herpes simplex virus type 1 envelope proteins to entry by endocytosis 2
Tri Komala Saria, Suzanne M. Pritchard
a, Cristina W. Cunha
a,b, George A. Wudiri
a,c, Elizabeth I. 3
Lawsa, Hector C. Aguilar
c, Naomi S. Taus
b, and Anthony V. Nicola
a* 4
aDepartment of Veterinary Microbiology and Pathology
, Washington State University,
bAnimal 5
Disease Research Unit, USDA-Agricultural Research Service, and cPaul G. Allen School for 6
Global Animal Health, Washington State University, Pullman, Washington, USA 7
8
9
RUNNING TITLE: HSV ENVELOPE PROTEINS AND ENDOCYTIC ENTRY 10
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12
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14
* Corresponding author. Mailing address: 15
Department of Veterinary Microbiology and Pathology 16
College of Veterinary Medicine 17
Washington State University 18
Pullman, WA 99164-7040 19
Phone: (509) 335-6003 20
Fax: (509) 335-8529 21
Email: [email protected] 22
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JVI Accepts, published online ahead of print on 9 October 2013J. Virol. doi:10.1128/JVI.02500-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.
2
ABSTRACT 24
Herpes simplex virus (HSV) proteins specifically required for endocytic entry but not direct 25
penetration have not been identified. HSVs deleted of gE, gG, gI, gJ, gM, UL45, or Us9 entered 26
cells via either pH-dependent or pH-independent endocytosis and were inactivated by mildly 27
acidic pH. Thus the required HSV glycoproteins, gB, gD, and gH-gL, may be sufficient for 28
entry regardless of entry route taken. This may be distinct from entry mechanisms employed by 29
other human herpesviruses. 30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
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Herpes simplex virus (HSV) has complex entry mechanisms and can utilize at least three 46
distinct cellular pathways: pH-independent fusion at the plasma membrane, or endocytosis, 47
which can be either pH-independent or pH-dependent. For example, entry into Chinese hamster 48
ovary (CHO) cells expressing nectin-1, human keratinocytes, and HeLa cells is through pH-49
dependent endocytosis (38, 39). HSV infects mouse melanoma cells expressing the nectin-1 50
receptor (B78C10) via pH-independent endocytosis (36). Infection of neurons and Vero cells 51
occurs via pH-independent fusion at the plasma membrane (29, 33, 38, 39, 46, 57). 52
How HSV chooses its pathway remains an open question. Both viral and cellular 53
determinants contribute to the selection of the entry pathway. Different strains of HSV-1 can 54
enter the same cell type via different pathways. In addition, different host receptors in the same 55
cell type can direct HSV to different pathways (12). Studies in various cell backgrounds have 56
indicated that cellular receptors such as nectin-1, nectin-2, PILRα, and integrins can influence 57
the entry route (1, 12, 25, 26, 36, 42, 48). This study tests the hypothesis that one or more 58
envelope proteins direct HSV to an endocytic entry route. For HSV, gB, gD, and the 59
heterodimer gH-gL are required via either the pH-independent or pH-dependent pathways (6, 20, 60
30, 40, 43, 57). Envelope proteins that are required for replication in Vero cells have been 61
deemed “essential” and those that are dispensable are termed “non-essential” (5, 31, 32, 56). 62
With the exception of gC and UL45p (15, 40), the contribution of the "non-essential" envelope 63
proteins to the endocytic entry process has not been evaluated. 64
The human herpesviruses Epstein-Barr virus (EBV) and human cytomegalovirus 65
(HCMV) each use distinct but overlapping glycoprotein complexes to enter cells via endocytic or 66
non-endocytic mechanisms (52-55). We explored the possibility that HSV might use a specific 67
4
envelope protein together with the required complex of gB, gD, and gH-gL to selectively 68
mediate entry via an endocytic pathway. 69
To determine how HSV-1 infects cells via endocytic entry, equivalent inocula of a panel 70
of membrane protein deletion-viruses (Table 1) were added to B78C10 (B78-nectin-1) cells (34). 71
Infectivity in Vero cells as measured by plaque formation was set to 100%. Wild type HSV-1 72
strains KOS and F had reduced plaquing efficiency on B78-receptor cells (Fig. 1) as reported 73
previously (34). As compared to wild type HSV F, each HSV mutant tested infected and formed 74
plaques on B78-nectin-1 cells (Fig. 1) indicating that gE, gG, gI, gJ, gM, UL45 and Us9 are 75
dispensable for infectivity in this cell type. HSV-1 UL20− and gK
− mutants were defective in 76
plaque formation on both cell types tested, forming microscopic, sparse clusters of infected cells 77
that were refractive to quantitation (data not shown), as reported previously in Vero cells (23, 78
27). In both cell types, gE− and gI
− HSVs produced smaller plaques relative to wild type HSV 79
(data not shown), consistent with the important role of the gE-gI complex in cell-to-cell spread of 80
HSV (13). 81
To examine entry more specifically, cell-free, extracellular preparations of the panel of 82
HSV mutants were evaluated by the beta-galactosidase reporter assay at 7 hr post-infection. 83
Virus was added to B78-nectin-1 cells or CHO-nectin-1 cells (24), both of which contain the E. 84
coli lacZ gene under the control of the HSV ICP4 gene promoter. Since gD is essential for entry 85
into all cell types due to its receptor-binding role (8, 17, 28, 40), HSV-1 gD-null virus was 86
included as a control to demonstrate the assay readout for a virus that is incapable of entry. Entry 87
of HSV-1 gD− in both B78-nectin-1 and CHO-nectin-1 cells was severely defective (Fig. 2A and 88
2B). The other mutant HSVs tested were each capable of entering B78-nectin-1 cells, ranging 89
from 0 to 5 times more effectively than wild type HSV (Fig. 2A). HSV gM− displayed enhanced 90
5
entry relative to wild type for reasons that are not apparent. For comparison, HSV-1 gD− entered 91
25 to 50 times less efficiently than wild type HSV (Fig. 2A). Each of the HSV mutants lacking 92
"non-essential" proteins also entered CHO-nectin-1 cells (Fig. 2B). Entry levels ranged from 5 93
times less than wild type to 20 times greater. It is not clear why the Us9− virus entered CHO-94
nectin-1 cells 5 times less effectively than wild type HSV. HSV-1 gD−
entered CHO-nectin-1 95
cells 63 to 333 times less effectively than wild type (Fig. 2B). Together with previous results 96
(15), this suggests that envelope proteins gE, gG, gI, gJ, gM, UL45p, and Us9p are individually 97
dispensable for entry into two cell lines that support endocytic entry. The possibility that one or 98
more "non-essential" proteins affect the rate of entry by endocytosis pathways cannot be ruled 99
out. It is also of interest whether deletion of two or more gene products might result in a serious 100
defect in entry. 101
Wild type HSV enters CHO-nectin-1 cells via a low pH-dependent endocytic pathway 102
(39). This pathway is thought to be critical for HSV infection of human keratinocytes (38). HSV 103
determinants of the pH-dependent entry pathway are not known. Although the panel of mutants 104
entered CHO-nectin-1 cells (Fig. 2B), it is possible that they bypassed the requirement for 105
intracellular low pH. To address this, cells were treated with ammonium chloride, which 106
elevates the normally low pH of endosomes and blocks HSV entry (39). Entry of gE−, gG
−, gI
−, 107
gJ−, gM
−, and Us9
− mutants was blocked by ammonium chloride in a concentration-dependent 108
manner similar to wild type HSV (Fig. 3). Together with previous results for a UL45− HSV (15), 109
this suggests that gE, gG, gI, gJ, gM, UL45p, and Us9 are not determinants of the pH-dependent 110
entry pathway taken by HSV. The core complex of gB, gD, and gH-gL may be sufficient for 111
entry regardless of cell type or entry route. 112
Inactivation of virions by pretreatment with low pH is a hallmark of viruses that enter via 113
6
an acid-dependent mechanism. The infectivity of HSV particles exposed to low pH is 114
irreversibly reduced (39). Virion gB undergoes pH-dependent conformational changes that are 115
reversible (7, 14, 16, 45). Thus, the target of acid-inactivation on the HSV particle is not clear. 116
To address this, gE−, gG
−, gI
−, gJ
−, gM
−, or Us9
− virions were treated with pH 7.2, 6.0, or 5.0. 117
Virions were neutralized back to pH 7.2 and added to Vero cells (39). The infectivity of each 118
mutant virus as measured by plaque assay was reduced by pretreatment with pH 6.0 or 5.0 in a 119
manner similar to wild type HSV (Fig. 4). Together with previous results (15), this suggests that 120
gE, gG, gI, gJ, gM, UL45, and Us9 do not determine the sensitivity of HSV to acid inactivation. 121
The target of low pH inactivation may be one or more components of the required entry 122
machinery, gB, gD, and gH-gL. 123
It is increasingly appreciated that herpesviruses traverse endocytosis pathways for 124
infectious entry. We demonstrate that HSV-1 gE, gG, gI, gJ, gM, UL45, and Us9 are non-125
essential for entry into cells that support either pH-dependent or pH-independent endocytic entry. 126
These results may be applicable to human epithelial cells that support endocytic entry of HSV. 127
Although the entry of several mutant viruses varied from wild type, none were as defective as 128
HSV gD−, which is negative for entry into all cell types tested. Although Us9p is not required 129
for pH-independent, endocytic entry, it may possibly influence pH-dependent entry in a direct or 130
indirect manner. For example, Us9 may directly impact pH-activation of gB or its absence may 131
adversely affect the incorporation of other viral proteins into the envelope. It is not known why 132
HSV-1 gE− and gI
− enter CHO-nectin-1 cells better than wild type HSV-1 or why HSV gM
− 133
enters B78-nectin-1 cells better than wild type. One possibility is that these glycoproteins may 134
have inhibitory effects on a given endocytic pathway. It will be of interest to determine whether 135
the lack of individual proteins alters the kinetics of entry into different types of cells. 136
7
Extracellular gK− and UL20
− HSVs were not obtained in sufficient quantities compared 137
to the other viruses tested in this study. This is due to the critical roles of gK and UL20 in HSV 138
egress (4, 21, 22, 27). However, cell-associated preparations of gK− and UL20
− viruses behaved 139
similarly to wild type HSV in all assays (data not shown). The roles of other envelope-140
associated proteins, including UL43, UL49.5, and UL56, in HSV entry remain to be determined. 141
The envelope proteins in this study are dispensable for entry via endocytic and non-endocytic 142
pathways. 143
EBV enters B cells by endocytosis and epithelial cells by direct penetration. EBV gp42 144
is required for fusion and entry in B cells but not epithelial cells (9, 35, 37, 54, 55). In a similar 145
vein, HCMV enters epithelial and endothelial cells by pH-dependent endocytosis and fibroblasts 146
by a pH-independent mechanism. HCMV UL128, UL130 and UL131 proteins are specifically 147
needed for epithelial and endothelial cell entry but not fibroblast entry (44, 50, 52, 53). 148
Homologs of EBV gp42 and HCMV UL128/130/131 are only present in the gammaherpesvirus 149
and betaherpesvirus subfamilies, respectively. HSV gE, gG, gI, gJ, gK, UL20, UL45, and Us9 150
are specific to the alphaherpesviruses, and our results suggest they do not play a role in 151
determining tropism in vitro. These proteins may play a more important role in entry in vivo. 152
HSVs deleted for gE, gG, gI, gJ and gK are non-essential for replication in mouse models of 153
HSV infection (5, 10). Both viral and cellular determinants affect HSV entry route. A specific 154
domain(s) on the required HSV entry complex of gB, gD, gH-gL may selectively direct HSV to 155
one pathway. Such a domain may interact with a region on another viral protein or interact with a 156
host determinant such as endosomal low pH or a cellular receptor ultimately leading to pathway-157
specific entry. 158
159
8
ACKNOWLEDGMENTS 160
This investigation was supported by Public Health Service grant AI096103 from the 161
National Institute of Allergy and Infectious Diseases (A. V. N) and a fellowship from the 162
Fulbright Scholar Program (T. K.). We are grateful to Joel Baines, Curtis Brandt, Gary Cohen, 163
Roselyn Eisenberg, Keith Jerome, David Johnson, Gus Kousoulas, Amy Sears and Patricia Spear 164
for generous gifts of reagents. We also thank Rich Scott and Bagus A. W. Sumohardjono for 165
help with figure preparation. 166
167
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327
328
329
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FIGURE LEGENDS 330
Figure 1. Envelope proteins gE, gG, gI, gJ, gM, UL45, and Us9 are dispensable for HSV 331
infectivity via endocytosis. B78-nectin-1 or Vero cells were infected with equivalent inocula of 332
wild type (F or KOS) or indicated mutant HSVs for 18-24 h at 370C. Titers were determined by 333
plaque assay with anti-HSV polyclonal antibody HR50 (Fitzgerald Industries, Concord, Mass). 334
Titers on Vero cells were set to 100%. Each experiment was performed in quadruplicate. Data 335
shown are the average of three experiments with standard deviation. None of the mutant virus 336
entry by endocytosis was statistically different from wild type as determined by Student's t-test. 337
338
Figure 2. Deletion mutant HSVs enter cells that support either pH-independent or pH-339
dependent endocytosis. B78-nectin-1 cells, which support pH-independent endocytosis (A) or 340
CHO-nectin-1 cells, which support pH-dependent endocytosis (B) were infected with equivalent 341
genome copy numbers of wild type (F or KOS) or mutant HSVs for 7 h at 370C. 1 x 10
6 genome 342
copies corresponded to MOIs ranging from 0.4 to 7. Beta galactosidase activity is an indication 343
of entry. The activity of wild type (F or KOS) was set to 1. Data shown are representative of 344
three independent experiments performed in quadruplicate. Error bars indicate standard 345
deviation. Entry of gD− and gM
− HSVs into B78-nectin-1 cells and entry of gD
−, gE
−, gI
− and 346
Us9− HSVs into CHO-nectin-1 cells were statistically different than wild type (p < 0.0001, 347
Student's t-test). 348
349
Figure 3. HSV-1 envelope proteins gE, gG, gI, gJ, gM, and Us9 are not viral determinants 350
of the pH-dependent entry pathway. CHO-nectin-1 cells were treated with the indicated 351
concentrations of ammonium chloride for 20 min at 370C. Wild type (F) or mutant HSVs was 352
13
added to cells (MOI of 1) for 7 h at 370C in the continued presence of agent. Entry was measured 353
as the percent of beta-galactosidase activity relative to that obtained in the absence of ammonium 354
chloride. Data are means of quadruplicate determinations with standard deviation. Data shown 355
are representative of three independent experiments. 356
357
Figure 4. Envelope proteins gE, gG, gI, gJ, gM, and Us9 do not determine HSV’s sensitivity 358
to low pH inactivation. Samples of wild type (F) or mutant HSVs (~ 100 PFU) were adjusted 359
to pH 7.2, 6.0 or 5.0, incubated at 37 °C for 10 min, and then neutralized to pH 7.2. Treated 360
virions were added to Vero cells, and plaque formation was measured as an indication of virus 361
entry and infection. The infectivity of each mutant HSV treated with pH 7.2 was defined as 362
100%. Data are the mean of quadruplicates with standard deviation. Data shown are 363
representative of three independent experiments. 364
365
366
14
Table 1. HSV-1s used in this study. 367
368
Virus name Parental
strain
Phenotype
and
designation in
this study
References
F - wild type (wt) (18)
KOS - wild type (wt) (2)
KOSgDく KOS gD- (11)
F-gE/GFP F gE- (19)
RAS104 F gG- (49)
F-gI/GFP F gI- (47)
F-gJ/GFP F gJ- (19)
R7216 F gM- (3)
UL45∆ KOS UL45- (51)
F-Us9/GFP F Us9- (41)
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
