key: cord-0959538-cntajl8x
authors: Lakshmi Narayan, Poornima Kotha; Kajon, Adriana E.
title: Human mastadenovirus-B (HAdV-B)-specific E3-CR1β and E3-CR1γ glycoproteins interact with each other and localize at the plasma membrane of non-polarized airway epithelial cells
date: 2020-04-15
journal: Virology
DOI: 10.1016/j.virol.2020.04.005
sha: 23b66f14689cb269037355cef15d24c1cb75530c
doc_id: 959538
cord_uid: cntajl8x

Abstract The E3 region of all simian and human types classified within species Human mastadenovirus B (HAdV-B) encodes two unique highly conserved ORFs of unknown function designated E3-CR1β and E3-CR1γ. We generated a HAdV-3 mutant encoding small epitope tags at the N-termini of both E3-CR1β and E3-CR1γ (HAdV-3 N-tag wt) and a double knock out (HAdV-3 N-tag DKO) mutant virus that does not express either protein. Our studies show that HAdV-3 E3-CR1β and E3-CR1γ are type I transmembrane proteins that are produced predominantly at late times post infection, are glycosylated, co-localize at the plasma membrane of non-polarized epithelial cells, and interact with each other. At their extreme C-termini HAdV-B E3-CR1β and E3-CR1γ possess a conserved di-leucine motif followed by a class II PDZ domain binding motif (PBM). HAdV-3 E3-CR1β and E3-CR1γ are dispensable for virus growth, progeny release, spread, and plaque formation in A549 cells.

Human adenoviruses (HAdV) encompass more than 90 genotypes that are classified into 7 39 species designated Human mastadenovirus A through G (HAdV-A through -G) (Espinola et al., 40 death protein/ADP) facilitates virus progeny release from infected cells at late stages of 83 infection. (Tollefson et al., 1996a; Tollefson et al., 1996b; Zou et al., 2004) . 84 The genomes of all simian and human types classified within species HAdV-B encode two 85 unique E3-CR1 genes designated CR1β and CR1γ which are believed to have arisen by gene 86 duplication (Signas et al., 1986) . As shown in Figure 1 , the genomes of some subspecies HAdV-87 B1 types encode a third highly polymorphic E3-CR1 gene designated CR1δ (Frietze et al., 2010; 88 Kajon et al., 2005) . These 3 genes have very low sequence similarity to any other known AdV 89 genes or to other sequences in the NCBI/GenBank database, making sequence-based prediction 90 of biological function challenging.

This paper presents the results of our initial characterization of the proteins encoded by the 92 HAdV-B specific E3-CR1β and E3-CR1γ genes using HAdV-3 as a model. 95 The sequences of E3-CR1β and E3-CR1γ ORFs encoded by various human and simian types 117 vectors 118 For our studies, we generated a HAdV-3 mutant virus encoding small epitope tags at the N-119 termini of E3-20.1K and E3-20.5K (HAdV-3 N-tag wt) and a double knock-out (DKO) mutant 120 virus that cannot express . For this purpose, a 121 fragment of the E3 region mapping between nucleotides 28000 and 29800 of the genome of (Table S1 ) that amplified the corresponding domains as shown in Figure S3 . 154 E3-20.1K and E3-20.5K class II PDZ binding motif (PBM) deletion mutants and the 155 corresponding di-leucine motif mutants (LL/AA) in which two alanines were substituted for the 156 two leucines ( Figure S3 ), were also generated using specific oligonucleotide primer sets that Scientific, Rockford, IL Cat# A32955)) and sonicated with five pulses of 22 kHz for 30 seconds. the TPL sequence at their 5' end. The constitutively expressed Rig/S15 was amplified as a 239 housekeeping control using the primers described above. The cycle number for amplification of 240 all CR1 genes and Rig/S15 was optimized empirically and set at 20 to fit within the exponential 241 range of amplification. The amplification products were analyzed by horizontal electrophoresis 242 on 1% agarose gels. Quantitative PCR (qPCR) was performed on the same set of cDNA samples 243 in a QuantStudio5 instrument (ThermoFisher Scientific, Rockford, IL) with PowerUp SYBR 244 Green Master Mix (ThermoFisher Scientific, Rockford, IL Cat# A25741) and the internal and 245 junction primer sets and the Rig/S15 primers described above. Each sample was tested in 246 duplicate. The data were normalized to Rig/S15. Fold changes in gene expressions relative to the 247 0 h time point were determined using the 2 -ΔΔCt method. were detected using two different sets of primers that distinguish early transcripts from MLP 314 transcripts. E3-20.1K and E3-20.5K transcripts were detectable as early as 6 hpi and were 315 abundantly produced at late times pi, as MLP transcripts carrying the TPL sequence at their 5' 316 end ( Figure 3A ). To validate these observations we performed quantitative PCR and readily 317 detected E3-20.1K and E3-20.5K MLP transcripts by 24 hpi ( Figure S5 ). Expression of both E3-318 20.1K and E3-20.5K proteins was detectable at 12 hpi, peaked at 48 hpi, and decreased by 72 hpi 319 ( Figure 3B ). Immunofluorescence microscopy studies revealed that at 24 hpi both E3-20.1K and 320 E3-20.5K predominantly co-localized to cytoplasmic vesicles and to a lesser extent at the plasma 321 membrane ( Figure 4A ). In an attempt to define the identity of the cytoplasmic vesicles, we performed co-localization studies using antibodies specific to PDI an endoplasmic reticulum 323 (ER) marker, Golgin-97, a trans-Golgi network (TGN) marker, EEA1, an early lysosome marker, 324 and LAMP2, a late endosome/lysosome marker. We observed that at 24 hpi both E3-20.1K and 325 E3-20.5K were present in the ER, TGN, and in early endosomes but not in late 

To confirm the predicted topology of HAdV-3 E3-E3-20.1K and E3-20.5K, we generated were detected at the plasma membrane of transfected cells irrespective of whether the cells had been permeabilized or not, while the C-tagged E3-20.1K and E3-20.5K proteins were detected 346 only when the cells were permeabilized ( Figure 5 ). 347 We next sought to investigate if any of the identified functional domains/motifs common to 348 E3-20.1K and E3-20.5K were required for the proper localization and orientation of these 349 proteins at the plasma membrane. We generated pMT2-PL or pMEGFP-C1 mammalian 362 The glycosylation state of E3-20.1K and E3-20.5K was examined in A549 cells infected RNA was extracted and reverse transcribed to cDNA. qPCR was performed with internal and 850 junction primers sets to detect E3-20.1K and E3-20.5K early and late transcripts. Samples were 851 assayed in duplicate and data were normalized to Rig/S15. A549 cells were infected with HAdV-3 wt, HAdV-3 N-tag wt, or HAdV-3 N-tag DKO mutant virus at a MOI of 1 pfu/cell. At indicated times post infection A) extracellular or B) total (intracellular and extracellular) infectious virus titers were determined by plaque assay to assess the kinetics of virus progeny release. Error bars represent the standard error of the mean from 3 biological replicates assayed in duplicate. C) A549 cells were infected with HAdV-3 wt, N-tag wt, or N-tag DKO mutant at various MOIs ranging from 10 to 0.001 pfu/cell. At 7 dpi, cells were fixed and stained to visualize virus dissemination. The data are representative of three independent experiments.

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CR1γ are type I transmembrane proteins • HAdV-B E3-CR1β and E3-CR1γ possess a C-terminal class II PDZ binding motif • HAdV-3 E3-CR1β and E3-CR1γ interact and co-localize at the plasma membrane • HAdV-3 E3-CR1β and E3-CR1γ are dispensable for virus progeny release and spread

1K and E3-20.5K proteins co-localize at the plasma membrane in non-polarized A549 cells. A549 cells were infected with the HAdV-3 N-tag wt virus at a MOI of 10 pfu/cell. At A) 24 and B) 48 hpi cell monolayers were fixed for immunofluorescence staining. E3-20.1K and E3-20.5K proteins were detected using primary antibodies specific to VSV-G and HA tags, respectively. Cells were imaged with a Zeiss LSM 8000 AiryScan confocal microscope using a 63X objective. Nuclei were stained with DAPI. Scale bar = 10µM. The images are representative of three independent experiments.

Non-permeabilized Permeabilized Non-permeabilized Permeabilized Non-permeabilized Permeabilized Non-permeabilized A B Figure 5 : HAdV-3 E3-20.1K and E3-20.5K are type I integral membrane proteins with N-luminal and C-cytoplasmic topology. pMT2-PL mammalian expression vectors encoding N-or C-terminally tagged A) E3-20.1K and B) E3-20.5K under the control of the HAdV-C2 derived major late promoter (MLP) were transfected into HEK 293T cells. At 72 hpt cell monolayers were fixed for immunofluorescence staining. During the staining procedure cells were either not permeabilized, or permeabilized with 0.5% Triton X-100 as indicated. The cells were stained with the primary antibody targeting the small epitope tag (green). Nuclei were stained with DAPI (blue). Images were acquired at 63x magnification using a Zeiss Axioskop epifluorescence microscope. TPL: Tri partite leader sequence. The images are representative of at least 20 fields analyzed from two independent experiments. were transfected into HEK 293T cells either individually or together. At 72 h post transfection, total cell lysates were analyzed by WB for expression of E3-20.1K and E3-20.5K with anti-tag antibodies. Additionally, the lysates were subjected to immunoprecipitation with anti VSV-G or anti HA conjugated magnetic beads to pull down E3-20.1K or E3-20.5K respectively. Pulled down samples were analyzed by WB for the presence of E3-20.5K or E3-20.1K, respectively. The blot is representative of three independent experiments.

Please check the following as appropriate: X All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version.X This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue.X The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript oThe following authors have affiliations with organizations with direct or indirect financial interest in the subject matter discussed in the manuscript:Author's name Affiliation