Full-length isoforms of KSHV LANA accumulate in the cytoplasm of cells undergoing the lytic cycle

85 Pages·2017·2.79 MB·English

JVI Accepted Manuscript Posted Online 4 October 2017
J. Virol. doi:10.1128/JVI.01532-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
1 Full-length isoforms of KSHV LANA accumulate in the cytoplasm of
2 cells undergoing the lytic cycle of replication
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4 H. Jacques Garriguesa, Kellie Howarda*, Serge Barcya, Minako Ikomaa, Ashlee V.
5 Mosesb, Gail H. Deutschc, David Wud, Keiji Uedae and Timothy M. Rosea,f# D
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7 aCenter for Global Infectious Disease Research, Seattle Children’s Research Institute, d
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8 Seattle, Washington, USA; bVaccine and Gene Therapy Institute, Oregon Health and f
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9 Science University, Beaverton, Oregon, USA; cPathology, Seattle Children’s Hospital,
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10 Seattle, Washington, USA; dDepartment of Laboratory Medicine, University of :
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11 Washington, Seattle, Washington, USA; eDivision of Virology, Department of i.a
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12 Microbiology and Immunology, Osaka University Graduate School of Medicine, .
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13 Osaka, Japan; fDepartment of Pediatrics, University of Washington, Seattle, /
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14 Washington, USA; A
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16 Running title: Cytoplasmic LANA 0
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18 #Address correspondence to Timothy M. Rose, [email protected] u
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19 *Present address: Covance, Redmond, WA, USA
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24 ABSTRACT
25 The latency-associated nuclear antigen (LANA) of the Kaposi sarcoma
26 herpesvirus (KSHV) performs a variety of functions to establish and maintain KSHV
27 latency. During latency, LANA localizes to discrete punctate spots in the nucleus
28 where it tethers viral episomes to cellular chromatin and interacts with nuclear
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29 components to regulate cellular and viral gene expression. Using highly sensitive o
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30 tyramide signal amplification (TSA), we determined that LANA localizes to the lo
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31 cytoplasm in different cell types undergoing the lytic cycle of replication after de d

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32 novo primary infection and after spontaneous, TPA-, or ORF50/RTA-induced m

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33 activation. We confirmed the presence of cytoplasmic LANA in a subset of cells in tt
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34 lytically-active multicentric Castleman disease lesions. The induction of cellular jv
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35 migration by scratch-wounding confluent cell cultures, culturing under subconfluent m
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36 conditions or induction of cell differentiation in primary cultures upregulated the r
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37 number of cells permissive for primary lytic KSHV infection. The induction of lytic n

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38 replication was characterized by high level expression of cytoplasmic LANA and r
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39 nuclear ORF59, a marker of lytic replication. Subcellular fractionation studies
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40 revealed the presence of multiple isoforms of LANA in the cytoplasm of 9

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41 ORF50/RTA-activated Vero cells undergoing primary infection. Mass spectrometry g
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42 analysis demonstrated that cytoplasmic LANA isoforms were full-length, containing s
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43 the N-terminal nuclear localization signal. These results suggest that trafficking of
44 LANA to different subcellular locations is a regulated phenomenon, which allows
45 LANA to interact with cellular components in different compartments during both
46 the latent and replicative stages of the KSHV lifecycle.
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47 IMPORTANCE
48 Kaposi sarcoma herpesvirus (KSHV) causes AIDS-related malignancies,
49 including lymphomas and Kaposi sarcoma. KSHV establishes life-long infections
50 using its latency-associated nuclear antigen (LANA). During latency, LANA localizes
51 to the nucleus where it connects viral and cellular DNA complexes and regulates
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52 gene expression allowing the virus to maintain long-term infections. Our research o
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53 shows that intact LANA traffics to the cytoplasm of cells undergoing permissive lytic lo
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54 infections and latently-infected cells in which the virus is induced to replicate. This d

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55 suggests that LANA plays important roles in the cytoplasm and nuclear m

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56 compartments of the cell during different stages of the KSHV life cycle. Determining tt
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57 cytoplasmic function and mechanism for regulation of the nuclear localization of jv
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58 LANA will enhance our understanding of the biology of this virus, leading to m
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59 therapeutic approaches to eliminate infection and block its pathological effects. r
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61 INTRODUCTION r
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62 Kaposi sarcoma herpesvirus (KSHV) is a gamma herpesvirus that causes
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63 several human malignancies, including Kaposi sarcoma (KS), primary effusion 9

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64 lymphoma (PEL), and AIDS-associated multicentric Castleman disease (MCD)(91, g
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65 104). Like all herpesviruses, KSHV has two distinct gene expression patterns that s
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66 result in either a persistent latent infection or an active lytic infection leading to
67 virus replication. However, latency is considered to be the default pathway for
68 KSHV infections. Initial studies showed that tumor cells in KS lesions were latently
69 infected as gene expression was very restricted, with two small RNAs, ie T0.7
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70 encoding K12 Kaposin and T1.1, a polyadenylated nuclear RNA (PAN), constituting
71 the bulk of KSHV transcripts (95, 116). Subsequent studies in KSHV-infected PEL
72 cell lines revealed the presence of a cluster of latency-associated transcripts
73 downstream of K12 encoding ORF 71 (v-FLIP), ORF 72 (v-cyclin), and ORF 73, the
74 latency associated nuclear antigen (LANA) (26, 47, 48, 99). Immunohistochemistry
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75 studies showed that LANA was present in the nucleus of KSHV-infected PEL cells as o
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76 well as the vast majority of spindled tumor cells in KS lesions (45, 48, 84). Anti- lo
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77 LANA antibodies gave a characteristic punctate staining pattern in the nucleus of d

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78 latently infected cells, which became a universal marker for KSHV latency. m

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79 A wide variety of endothelial, epithelial, fibroblast and lymphocyte cells are tt
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80 susceptible to KSHV infection resulting in a typical latent infection with restricted jv
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81 gene expression and accumulation of nuclear LANA (7, 8, 24, 87). KSHV infection of m
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82 endothelial and fibroblast cells induces an early transient expression of ORF50, the r
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83 replication transactivator (RTA), which is the only viral gene that is necessary and n

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84 sufficient for KSHV replication (66, 98). This is followed by a brief burst of r
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85 expression of a subset of lytic genes with immunomodulatory and apoptotic
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86 functions, whose transcripts disappear 8-24 hours post infection (53). It was 9

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87 suggested that the transient expression of this subset of genes was important for the g
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88 establishment and maintenance of latency, but did not lead to viral replication, as s
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89 genes critical for DNA replication, such as ORF9, the viral DNA polymerase, and
90 ORF59, the DNA polymerase processivity factor, were absent from the RTA-induced
91 transient gene expression burst. The early spike in RTA induces LANA expression,
92 which then shuts off RTA expression through transcriptional repression of the RTA
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93 promoter (24, 57, 64). LANA autoactivates its own promoter leading to the
94 accumulation of LANA in the nucleus and the establishment of latency in the
95 infected cell (56).
96 Very few examples of primary KSHV lytic infections have been observed
97 previously. Productive lytic infection has been documented in human endothelial
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98 cells and human papillomavirus 16-immortalized keratinocytes; however, this o
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99 required high titers of concentrated recombinant KSHV (32, 37). Primary tonsillar lo
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100 B-cells grown in a lymphoid aggregate culture were also productively infected by d

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101 KSHV (73). The majority of KSHV infection studies used chemical inducers such as m

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102 the phorbol ester TPA or sodium butyrate, an inhibitor of histone deacetylase, to tt
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103 reactivate latent KSHV infections and induce lytic replication (71, 88, 89). jv
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104 Additionally, overexpression of recombinant KSHV ORF50 RTA has been shown to m
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105 reactivate latent KSHV infections in a variety of cell types (67). The switch from r
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106 latent to lytic replication after induction is initiated by the immediate early n

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107 expression of RTA, which then activates the entire lytic replication cycle leading to r
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108 the expression of a cascade of viral genes in an ordered progression resulting in the
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109 production of infectious virions. 9

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110 Early on, it was noted that a small percentage of cells in latently-infected g
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111 cultures expressed markers of lytic KSHV replication. In endothelial cells for s
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112 example, ~1% of cells expressed ORF59 lytic marker forty-eight hours after de novo
113 infection, indicating “spontaneous” reactivation of the lytic program of replication
114 (55). Lytic cycle gene expression, including ORF59, ORF26 (major capsid protein),
115 and ORF K2 (vIL-6), has also been detected in a small percentage of cells in latently
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116 infected PEL cell cultures and in KS tumor lesions (3, 44, 78, 96). Because this lytic
117 gene expression is confined to a small proportion of cells in infected cultures, it has
118 been difficult to determine whether it is due to an active lytic infection in specific
119 cells or whether this represents an additional viral transcription program that could
120 be cell-type specific or associated with particular cellular proliferation and/or
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121 differentiation states. However, using a model of primary lytic replication, we have o
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122 shown a direct correlation between the expression of ORF59, the induction of the lo
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123 lytic program of replication and production of infectious virions of a primate d

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124 homolog of KSHV (9). Furthermore, previous studies have shown that an ORF59 m

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125 deletion mutant of KSHV is defective in virion production, substantiating the critical tt
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126 role of ORF59 in the lytic cycle of replication (70). jv
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127 We have observed that cell lines showing spontaneous reactivation of KSHV m
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128 after latent infection, such as HEK293 and Vero, have moderate constitutive levels of r
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129 cellular transcription factors that can directly activate the RTA promoter (24). This n

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130 level of promoter activity is not sufficient to drive RTA-induced lytic gene r
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131 expression and viral replication in the majority of infected cells. However, the
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132 latently-infected cells are poised to allow reactivation of the viral genome and 9

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133 induction of viral replication, and small changes in the status of the cell can induce g
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134 spontaneous reactivation. Treatment with phorbol esters or sodium butyrate s
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135 increases the level of cellular transcription factors activating the RTA promoter,
136 resulting in increased RTA expression and induction of viral lytic replication. In
137 contrast, cell lines such as the gastric epithelial cell line (AGS) have minimal
138 constitutive expression of cellular factors that can activate the RTA promoter, and
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139 KSHV-infected AGS cells exhibit a deep latent state, with no evidence of
140 spontaneous reactivation (24).
141 Reactivation of lytic replication by cellular differentiation has been studied in
142 oral epithelial cells infected with the recombinant rKSHV.219 virus, which expresses
143 green fluorescent protein (GFP) from a constitutive cellular EF-1 promoter during
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144 typical latent infections and red fluorescent protein (RFP) from the lytic cycle PAN
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145 promoter after reactivation (102). Culturing of human oral keratinocytes (HOK) lo
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146 latently infected with rKSHV.219 in organotypic raft cultures induced differentiation d

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147 of infected cells at the apical surface of the stratified epithelial layer, as evidenced by m

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148 increased expression of the differentiation antigens, involucrin and keratins 6, 13, tt
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149 14 and 19. The differentiation of these epithelial cells induced expression of RFP jv
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150 and the late virion glycoprotein K8.1, as well as the production of infectious m
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151 rKSHV.219 at the epithelial surface (43). In further studies using a submerged rg
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152 direct-plating model of keratinocyte differentiation, differentiated rKSHV.219- n

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153 infected primary keratinocytes expressed RFP after plating in a suprabasal position r
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154 onto a confluent epithelial cell culture (93). These studies showed that 2
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155 differentiation and suprabasal positioning in an epithelial layer were sufficient to 9

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156 activate KSHV lytic replication. g
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157 In contrast to the latency observed in KS lesions and PEL tumors, KSHV- s
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158 associated MCD lesions show a high percentage of infected cells expressing early,
159 intermediate and late-stage KSHV proteins, including ORF59, K2 (vIL-6), K8 (K-
160 bZip), K9 (vIRF-1), K10 (vIRF-4) and the K8.1 glycoprotein (1, 44, 78, 79). The
161 elevated expression of KSHV lytic genes suggests that MCD lesions are sites of lytic
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162 replication where differentiation signals in a unique B cell subset activate the lytic
163 program of KSHV transcription.
164 In latent KSHV infections, LANA localizes to punctate foci in the nucleus at
165 sites where LANA tethers viral episomes to host chromatin, ensuring partition of the
166 KSHV genomes to daughter nuclei during mitosis (5, 19). In addition to episome
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167 maintenance, LANA regulates viral and cellular gene transcription (2, 25, 34, 56, 60), o
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168 recruits enzymes that affect chromatin remodeling (41, 50, 94), promotes cell lo
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169 survival and growth (11, 27, 63, 65, 101, 104) and inhibits apoptosis (29, 46). LANA d

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170 also represses the expression of RTA and other KSHV lytic genes and inhibits the m

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171 KSHV replication program (57, 58, 101). tt
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172 LANA is a large protein with three structural domains (90). The serine- jv
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173 proline-rich N-terminus contains a complex bipartite nuclear localization signal, m
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174 which utilizes both classical and non-classical pathways for nuclear import (18, 81), r
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175 a chromatin-binding motif (CBM)(106), and domains responsible for interaction n

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176 with various transcription regulators (54, 101). The C-terminal domain is r
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177 responsible for binding to the terminal repeats of the KSHV genome and tethering
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178 the viral episome to host cell chromatin (5). This domain also interacts with various 9

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179 cellular proteins that localize to the nucleus, including p53, pRb and MeCP2 (29, 69, g
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180 83), and nuclear localization is observed when the LANA is expressed as a truncated s
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181 C-terminal peptide (81, 92). The central region of LANA contains several repetitive
182 acidic regions whose heterogeneity across different KSHV strains results in LANA
183 proteins ranging from 1003 to 1162 amino acids in length. Whereas the predicted
184 size of LANA is ~100-120 Kd, the protein migrates as a number of different forms in
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185 SDS gels with mobilities from 130-234 kDa (48, 84). LANA variants have been
186 associated with non-canonical translation initiation (100), non-canonical
187 polyadenylation (14), and caspase modification (20).
188 In early immunohistochemical studies, LANA staining was detected in the
189 cytoplasm of a subset of cells in MCD lesions (1, 74). Subsequently, isoforms of
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190 LANA were detected in cytoplasmic extracts of KSHV-infected HEK293 and BCBL-1 o
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191 PEL cells (100, 115). Induction of BCBL-1 cells with TPA increased the levels of lo
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192 cytoplasmic LANA isoforms (115), and cytoplasmic forms bound to the innate d

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193 immune sensor cGMP-AMP synthase (cGAS) and to components of the MRN (Mre11- m

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194 Rad50-NBS1) repair complex, suggesting a role in antagonizing the innate immune tt
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195 response and thus facilitating lytic reactivation (68, 115). We previously developed jv
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196 ultra-sensitive enhancement of fluorescent labeling for confocal microscopy using m
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197 tyramide signal amplification (TSA) (35, 36). During these studies, we observed r
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198 strong LANA fluorescence in the cytoplasm of specific cells in different KSHV- n

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199 infected cultures using a LANA-specific monoclonal antibody. In the current study, r
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200 we have used TSA-enhanced confocal microscopy and other techniques to
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201 investigate the connection between cytoplasmic LANA isoforms and KSHV lytic 9

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202 replication. We confirmed the presence of cytoplasmic LANA in lytically-active MCD g
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203 lesions. In addition, we observed a strong correlation between the expression of s
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204 cytoplasmic LANA and nuclear ORF59 after lytic reactivation of KSHV in latently
205 infected cells as well as after primary lytic infections of KSHV in differentiated
206 and/or migrating cells. Subcellular fractionation revealed the presence of multiple
207 LANA isoforms in the cytosol, and full-length isoforms containing the N-terminal
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208 nuclear localization were detected by mass spectrometry. The presence of full-
209 length isoforms in the cytosol is suggestive of a regulatory process that can block or
210 enhance nuclear localization of LANA, thereby altering the cellular distribution of
211 this critical viral protein during the different lytic and latent programs of KSHV
212 infection.
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214 RESULTS lo
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215 Cytoplasmic LANA in KSHV-associated MCD lesions. Previously, two d

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216 different staining patterns were reported for antibodies to KSHV ORF73 LANA in m

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217 lymph nodes from patients with MCD. One pattern was similar to that seen in tt
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218 spindled tumor cells in KS lesions with the presence of multiple punctate dots in the jv
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219 nucleus. The other pattern showed diffuse staining in both the cytoplasm and m
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220 nucleus (1, 74). To confirm this differential localization, we prepared paraffin- r
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221 embedded sections from a KS skin nodule and two MCD lymph nodes for KSHV n

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222 LANA localization using monoclonal antibody LN53 (49). In the KS lesion, LN53 r
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223 specifically localized to nuclei in KS spindle cells where the brown
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224 diaminobenzidine chromogen formed punctate LANA dots (Fig 1A; arrowheads). In 9

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225 MCD lesions, the LN53 antibody gave two different staining patterns. In some cells, g
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226 LN53 staining was confined to the nucleus with punctate structures, similar to the s
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227 pattern seen in KS lesions (Fig 1B; arrowheads). In other cells, the LN53 also
228 localized diffusely throughout the cytoplasm (Fig 1B; arrows). The cytoplasmic
229 staining was confined to individual cells having distinct borders, and thus was not
230 due to nonspecific chromogen diffusion. The cells with cytoplasmic LANA staining
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