Facultad de Ciencias
Departamento de Biología Molecular
Contribution of herpes simplex virus
glycoprotein G to viral pathogenesis
Alberto Domingo López Muñoz
Madrid, 2019
Facultad de Ciencias
Departamento de Biología Molecular
Programa de Doctorado en Biociencias Moleculares RD99/2011
Contribution of herpes simplex virus glycoprotein G to viral pathogenesis
Contribución de la glicoproteína G del virus herpes simplex a la patogénesis viral
Memoria presentada para optar al grado de Doctor en Biociencias Moleculares
Alberto Domingo López Muñoz Licenciado en Biología
Directores de Tesis:
Dr. Antonio Alcamí Pertejo Dr. Alberto Rastrojo Lastras
Tutor:
Dr. Manuel Fresno Escudero
Este trabajo ha sido realizado en el Centro de Biología Molecular
Severo Ochoa (CSIC-UAM)
Index
Preface ... v
List of abbreviations ... vii
List of figures, tables and movies ... xi
1. Abstract / Resumen ... 1
2. Introduction ... 7
2.1. HUMAN ALPHAHERPESVIRUSES ... 9
2.2. HSV BIOLOGY ... 10
2.2.1. Life cycle ... 10
2.2.2. Lytic stage ... 10
2.2.3. Virion and genome structure ... 12
2.2.4. Serotype-dependent neurotropism ... 12
2.3. GENOMIC AND VIRULENCE VARIABILITY ... 14
2.3.1. Replication and recombination of the viral genome ... 14
2.3.2. Generation of genomic/genetic variability ... 15
2.3.3. Viral isolates, strains and consensus genomes ... 16
2.4. TOOLS AND MODELS FOR HSV STUDIES ... 17
2.4.1. Systems to generate recombinant HSVs ... 17
2.4.2. Murine models of HSV pathogenesis and latency ... 19
2.5. HOST IMMUNE RESPONSE AND HSV EVASION ... 21
2.5.1. Countermeasures against innate and adaptive immunity ... 21
2.5.2. Role of chemokines (CK) in HSV infection ... 22
2.5.3. Viral CK-binding proteins (vCKBPs) encoded by herpesviruses ... 23
2.5.4. HSV gG ... 24
3. Objectives ... 27
4. Material and methods ... 31
4.1. Cell lines ... 33
4.2. Ethical statement ... 33
4.3. Viruses ... 33
4.4. Production of viral stocks ... 33
4.5. Viral DNA preparation and purification ... 34
4.6. Construction and sequencing of Illumina libraries ... 34
4.7. Construction and sequencing of Pacific Biosciences (PacBio)
libraries ... 34
4.8. Alignments of Illumina and PacBio sequencing data ... 35
4.9. Assembly and annotation of HSV-1 (SC16), HSV-2 (333) and HSV-2 (MS) genomes ... 35
4.10. Genetic variant analysis ... 36
4.11. Selection of HSV viral clones and genetic variability studies ... 36
4.12. i.n. and i.v. mouse models of infection ... 36
4.13. Plasmids construction ... 37
4.14. gRNA design and cloning ... 40
4.15. Method for generation of mutant HSVs by CRISPR/Cas9 system ... 41
4.16. Generation of HSV UL26-27 Red ... 43
4.17. Generation of HSV Red ∆gG-eGFP ... 43
4.18. Generation of the HSV gG mutant collection ... 43
4.19. Western blot antibodies ... 46
4.20. Replication kinetics curves ... 46
4.21. Skin mouse model of infection ... 46
4.22. Intravital multiphoton microscopy (MPM) imaging and analysis ... 48
4.23. Flow cytometric analysis ... 48
4.24. qPCR analysis ... 48
4.25. Corneal scarification model of infection and latency-reactivation .... 49
4.26. Statistical analysis ... 49
4.27. Data availability ... 50
5. Results ... 51
5.1. GENOMIC CHARACTERIZATION OF HSV STRAINS... 53
5.1.1. Genome sequencing, assembly and variability of HSV strains ... 53
5.1.1.1. HSV-1 strain SC16 ... 53
5.1.1.2. HSV-2 strain 333 ... 53
5.1.2. Selection and characterization of plaque-purified viral candidates ... 54
5.1.2.1. Sequencing and analysis of five viral clones isolated from original HSV stocks ... 54
5.1.2.2. Deeper sequencing and analysis of selected candidates from original HSV stocks ... 57
5.1.2.3. In vivo testing of selected viral clones against original viral stocks ... 60
5.1.3. Generation of HSV genetic variability in cell culture ... 60
Index 5.2. BAC AND CRISPR/CAS9: LOOKING FOR A SYSTEM TO GENERATE
RECOMBINANT HSVs ... 65
5.2.1. The BAC system ... 67
5.2.1.1. Sequencing and analysis of BAC-derived HSVs ... 67
5.2.1.2. In vivo testing of BAC-derived HSVs ... 71
5.2.2. The CRISPR/Cas9 system ... 72
5.2.2.1. Strategy ... 72
5.2.2.2. Design and optimization ... 72
5.2.2.3. Quantification of efficiency ... 76
5.3. CHARACTERIZATION OF THE HSV gG MUTANT COLLECTION ... 76
5.3.1. Sequencing and analysis of candidates ... 78
5.3.2. Characterization of recombinant HSVs: gG expression and replication kinetics ... 78
5.3.3. In vivo testing of the UL26-27 Red marker insertion ... 83
5.4. IN VIVO CONTRIBUTION OF gG TO HSV INFECTION ... 84
5.4.1. Mouse model of infection for cell recruitment studies ... 84
5.4.1.1. Replication kinetics and visualization of epicutaneous HSV infection . 84 5.4.1.2. Innate immune cell recruitment triggered by HSV infection ... 86
5.4.2. Mouse models of infection for pathogenesis, latency and reactivation studies ... 90
5.4.2.1. gG contribution to viral pathogenesis ... 91
5.4.2.2. Colonization of CNS tissues, latency and reactivation ... 93
6. Discussion ... 97
6.1. HSV DIVERSITY AND EVOLUTION ... 99
6.1.1. Generation of genetic variability in cell culture ... 99
6.1.2. Disclosing viral diversity through NGS technologies ... 101
6.1.3. Future perspectives ... 102
6.2. GENOME EDITING OF dsDNA VIRUSES ... 102
6.2.1. Making recombinant viruses in the CRISPR/Cas9 era ... 103
6.2.2. Optimizing CRISPR/Cas9 to generate mutant dsDNA virus collections . 104 6.3. ROLE OF gG IN HSV INFECTION ... 105
6.3.1. Contribution to viral pathogenesis ... 107
6.3.2. Contribution to latency establishment and subtype-dependent neurotropism ... 108
7. Conclusions / Conclusiones ...113
8. References ...119
9. Supplemental information ... 141
9.1. Supplementary figures ...143
9.2. Supplementary movies ...157
9.3. Supplementary tables ...161
9.4. Scientific publications ...303
Preface
This work describes the contribution of herpes simplex virus (HSV) glycoprotein G to viral pathogenesis and neurotropism in different contexts and models of infection. Genetic analysis of different HSV laboratory strains, as well as strategies for generating collections of recombinant viruses will also be presented. The reader will be provide with an overview of HSV biological features and clinical relevance, followed by a discussion of the results obtained over the course of this project. This dissertation was submitted to the Universidad Autónoma de Madrid (UAM), Spain, to obtain the PhD degree.
The experimental work discussed here was carried out between October 2014 and October 2018 at Centro de Biología Molcular Severo Ochoa (CSIC-UAM, Madrid, Spain), under the supervision of Dr.
Antonio Alcamí Pertejo and Dr. Alberto Rastrojo Lastras (Spanish Research Council, CSIC).
Time-lapse microscopy training was received during a research stay at the Imperial College London (UK), under the supervision of Prof. Peter O’Hare. The results presented in regard to the mouse skin model of infection and intravital microscopy analyses were carried out during a second stay at the US National Institute of Allergy and Infectious Diseases (Maryland, USA), under the supervision of Dr. Jonathan Yewdell and Dr. Heather Hickman.
The project was funded by the Spanish Ministry of Science, Innovation and Universities (Grants SAF2012-38957 and SAF2015-67485-R). The Spanish Ministry of Education provided a FPU PhD studentship in support of this project, obtained in national competition (FPU13/05425). International research stays were financially supported by the Sir William Dunn School of Pathology (University of Oxford, UK), the Federation of European Microbiological Societies (FEMS) and the European Molecular Biology Organization (EMBO).
Este trabajo describe la contribución de la glicoproteína G del virus herpes simplex virus (HSV) a la patogénesis viral y el neurotropismo en diferentes contextos y modelos de infección. El análisis genético de diferentes cepas de laboratorio, así como estrategias para generar colecciones de virus recombinantes también serán presentadas. El lector recibirá una visión general de las características biológicas y la relevancia clínica de HSV, seguido de una discusión de los resultados obtenidos durante el curso de este proyecto. Esta disertación fue presentada en la Universidad Autónoma de Madrid (UAM), en España, para obtener el grado de Philosophiæ doctor.
El trabajo experimental discutido fue desarrollado entre octubre de 2014 y octubre de 2018 en el Centro de Biología Molecular Severo Ochoa (CSIC-UAM, Madrid, España), bajo la supervisión del Dr. Antonio Alcamí Pertejo y del Dr. Alberto Rastrojo Lastras (Consejo Superior de Investigaciones Científicas, CSIC).
Se recibió entrenamiento en microscopía de lapso de tiempo durante una estancia de investigación en el Imperial College London (Reino Unido), bajo la supervisión del Prof. Peter O’Hare. Los resultados presentados con respecto al modelo de infección de piel de ratón y los análisis de microscopía intravital fueron llevados a cabo durante una segunda estancia en el US National Institute of Allergy and Infectious Diseases (Maryland, EE. UU.), bajo la supervisión del Dr. Jonathan Yewdell y la Dra.
Heather Hickman.
Este proyecto fue financiado por el Ministerio de Ciencia, Innovación y Universidades (Proyectos SAF2012-38957 and SAF2015-67485-R). El Ministerio de Educación proporcionó un contrato FPU como apoyo a este proyecto, obtenido en competición nacional (FPU13/05425). Las estancias de investigación internacionales fueron financiadas por la Sir William Dunn School of Pathology (Universidad de Oxford, Reino Unido), la Federación Europea de Sociedades de Microbiología (FEMS) y la Organización Europea de Biología Molecular (EMBO).
List of abbreviations
BAC Bacterial artificial chromosome
bp Base pairs
Cas9 CRISPR-associated protein 9 CAV Cell-associated virus
CDS Coding sequence
CGRP Calcitonin gene related peptide
CK Chemokine
CMV Citomegalovirus
CNS Central nervous system
CR Coding region
CRISPR Clustered regularly interspaced short palindromic repeats
DC Dendritic cell
DMEMs Dulbecco’s Modified Eagle’s Medium supplemented with …
dpe Days post-explant
dpi Days post-infection DRG Dorsal root ganglia
DSB Double-strand break
dsDNA Double-stranded DNA
eGFP Enhanced green fluorescent protein EV Extracellular virus
FNE Free nerve ending
GAG Glycosaminoglycan
gB HSV glycoprotein B
gC HSV glycoprotein C
gD HSV glycoprotein D
GDNF Glial cell-derived neurotrophic factor
gE HSV glycoprotein E
gG HSV glycoprotein G
gG1 HSV-1 gG
gG2 HSV-2 gG
gI HSV glycoprotein I
GPCR G protein-coupled receptor
gRNA guide RNA
HDR Homology-directed repair HF High fidelity
hpi Hours post-infection
HSV Herpes simplex virus
HSV-1 HSV type 1
HSV-2 HSV type 2
i.n. Intranasal i.v. Intravaginal
IFN Interferon
InDels Insertions/deletions
IRL HSV genomic internal repeat long IRS HSV genomic internal repeat short
ISG IFN-stimulated gene
Kbp Kilobase pairs
KO Knock out
LAT Latency-associated transcript
mgG2 Mature gG2
miRNA Micro RNAs
MOI Multiplicity of infection MPM Multiphoton microscopy
NCR Non-coding region
ncRNA Non-coding RNA
NGF Nerve growth factor
NGS Next generation sequencing NHEJ Non-homologous end joining NK Natural killer
ORF Open reading frame
oriL HSV origin of DNA replication within the UL segment
oriS HSV origins of DNA replication within the IRS/TRS repeated regions PacBio Pacific Biosciences
PFU Plaque-forming unit
QF Quality-filtered ROI Regions of interest
SC Stop codon
SEM Standard error of the mean
SgG2 Secreted gG2
SNP Single nucleotide polymorphism SPF Specific pathogen-free
TG Trigeminal ganglia TLR Toll-like receptor
TrkA Tropomyosin receptor kinase A TRL HSV genomic terminal repeat long TRS HSV genomic terminal repeat short UL HSV unique long genomic segment
List of abbreviations US HSV unique short genomic segment
vCKBP Viral chemokine-binding protein
VgC VZV glycoprotein C
VZV Varicella zoster virus
List of figures, tables and movies
2. Introduction
Figure I1. Life cycle stages of HSV.
Figure I2. HSV virion and genomic structure.
Figure I3. Viral genome populations contain a broad range of variations which may change their frequencies over time.
Figure I4. Schematic of the CRISPR/Cas9 system involved in bacterial acquired immunity and their application in genome editing procedures.
Figure I5. vCKBP mechanisms of action.
Figure I6. HSV gG1 and gG2 structural and functional overview.
Table I1. HSV strains commonly used in experimental research.
Table I2. Different routes of inoculation for experimental studies of HSV animal models.
Table I3. Interaction and effects of vCKBPs encoded by herpesviruses described to date.
4. Material and methods
Figure M1. Schematic of the construction of donor plasmids.
Figure M2. gRNAs cloned and used in this thesis.
Figure M3. Flowchart of the 2 gRNAs / 2 progenies method used to generate CRISPR/Cas9- derived recombinant HSVs.
Figure M4. Schematic of recombinant HSVs UL26-27 Red generation.
Figure M5. Diagram for generation of the mutant HSV Red ∆gG-eGFP.
Figure M6. Representative generation of mutant HSVs gG-KO, giving as examples of the workflow followed to produce the rest of gG mutant HSVs.
5. Results
Figure R1. Schematic of the HSV-1 strain SC16 and HSV-2 strain 333 de novo assembled genomes.
Figure R2. Diagram of the workflow for selection and characterization of plaque-purified viral clones from HSV-1/2 original stocks.
Figure R3. Genomic variability analysis of HSV-1 viral clones.
Figure R4. Genomic variability analysis of HSV-2 viral clones.
Figure R5. Numbers of total and de novo mutations from variant analysis of HSV-1 and HSV-2 isolated clones 1-5.
Figure R6. Deep genetic variability analysis of HSV-1 isolated clones 2 and 3, and HSV-2 clones 1 and 5.
Figure R7. Pathogenesis of HSV-1 and HSV-2 isolated viral candidates against their corresponding original stocks.
Figure R8. Flowchart of HSV genomic variability studies.
Figure R9. Generation of HSV genetic variability in cell culture.
Figure R10. Collection design of HSV-1 and HSV-2 recombinant viruses in which gG is replaced by a mutant version or swapped by its HSV type orthologue.
Figure R11. Structural and variability analysis of HSV-1 strain 17 wt and BAC-derived viral genomes.
Figure R12. Structural and variability analysis of HSV-2 strain MS wt and BAC-derived viral genomes.
Figure R13. Pathogenesis of HSV-1 (17) and HSV-2 (MS) against their respective BAC-derived viruses.
Figure R14. Color-based selection strategy to generate CRISPR/Cas9-derived recombinant HSVs.
Figure R15. Detailed flowchart of the 2 gRNAs / 2 progenies method used to generate CRISPR/
Cas9-derived recombinant HSVs.
Figure R16. Efficiency quantification of the 2 gRNAs / 2 progenies method to generate CRISPR/
Cas9-derived HSVs.
Figure R17. Numbers of total mutations (upper panels) are filtered by non-conservative changes (middle and lower panels) and represented as columns, for each HSV-1 (blue) and HSV-2 (red) gG mutant viruses generated by the CRISPR/Cas9 system.
Figure R18. Expression of gG from recombinant viruses.
Figure R19. Replication kinetics of HSV-1 and HSV-2 mutant viruses in cell culture.
Figure R20. Virulence in animal models to assess the impact of the Red cassette insertion into the UL26-27 intergenic region in HSV-1 and HSV-2 (b).
Figure R21. HSV infection of the mouse skin.
Figure R22. Innate immune cell recruitment into murine skin triggered by HSV infection (I).
Figure R23. Innate immune cell recruitment into murine skin triggered by HSV infection (II).
Figure R24. Contribution of gG to HSV pathogenesis.
Figure R25. Contribution of gG to HSV neurovirulence, latency and reactivation.
Table R1. Sequencing statistics of HSV-1 and HSV-2 original stocks.
List of figures, tables and movies Table R2. Numbers of categorized mutations from variant analysis of HSV-1 and HSV-2 original
stocks sequencing data.
Table R3. Illumina sequencing statistics of HSV-1 and HSV-2 plaque-isolated clones.
Table R4. Numbers of categorized mutations from variant analysis of HSV-1 and HSV-2 plaque- isolated clones sequencing data.
Table R5. Illumina deep sequencing statistics of HSV-1 and HSV-2 candidate clones.
Table R6. Numbers of categorized mutations from variant analysis of HSV-1 and HSV-2 candidate clones deep sequencing data.
Table R7. Illumina deep sequencing statistics of HSV-1 and HSV-2 isolated clones after 5 (P5) and 10 (P10) passages in Vero and HaCaT cells.
Table R8. Numbers of categorized mutations from variant analysis of sequencing data originating from HSV-1 and HSV-2 isolated clones after 5 (P5) and 10 (P10) passages in Vero and HaCaT cells.
Table R9. Illumina and PacBio sequencing statistics of HSV-1 strain 17 wt, HSV-2 strain MS wt and their corresponding BAC-derived viruses.
Table R10. Numbers of categorized mutations after variant analysis of sequencing data from HSV-1 strain 17 wt, HSV-2 strain MS wt and their corresponding BAC-derived viruses.
Table R11. Illumina sequencing statistics of HSV-1 and HSV-2 recombinant viruses, including Red, ∆gG-eGFP and gG mutant viruses.
Table R12. Numbers of categorized mutations after variant analysis of sequencing data from HSV-1 and HSV-2 recombinant viruses, including Red, ∆gG-eGFP and gG mutant viruses.
Table R13. Survival rates after i.n. and i.v. infections with HSV-1/2 Red and gG mutants.
6. Discussion
Figure D1. Models of neuron-specific outcome of infection with HSV-1 and HSV-2 for each tissue, showing the contribution of gG1 and gG2 to each scenario of infection.
9. Supplemental information
Figure S1. Deep genetic variability analysis of HSV-1 plaque-purified clone 2, after 5 and 10 passages in Vero and HaCaT cells.
Figure S2. Deep genetic variability analysis of HSV-1 plaque-purified clone 3, after 5 and 10 passages in Vero and HaCaT cells.
Figure S3. Deep genetic variability analysis of HSV-2 plaque-purified clone 1, after 5 and 10 passages in Vero cells.
Figure S4. Deep genetic variability analysis of HSV-2 plaque-purified clone 5, after 5 and 10 passages in Vero and HaCaT cells.
Figure S5. Viral titers (mean ±SEM) per ear determined by plaque assay after infection of BALB/
cByJ mice with indicated viruses after 3 dpi.
Figure S6. Levels of the indicated viral genomes from infected ears of BALB/cByJ mice.
Figure S7. Representative (MPIs) of MPM z stack images of ventral ear sections from T-bet- eGFP mice and LysM-eGFP mice.
Figure S8. MPIs of MPM z stack images of ventral ear sections from T-bet-eGFP mice (green NK and CD4+ cells), within a HSV-1 Red lesion at 3 dpi (from Movie S1).
Figure S9. Flow dot plots of cells recovered from collagenase-dissociated ears.
Figure S10. Innate immune cell recruitment into murine skin triggered by HSV infection (III).
Figure S11. Representative pictures of reactivation from trigeminal ganglia explanted in Vero cell monolayers after 8 dpe.
Movie S1. MPIs of MPM z stack images of ventral ear sections from T-bet-eGFP mice infected with HSV-1 Red.
Movie S2. MPIs of MPM z stack images of ventral ear sections from T-bet-eGFP mice infected with HSV-1 Red gG-SC.
Movie S3. MPIs of MPM z stack images of ventral ear sections from T-bet-eGFP mice infected with HSV-1 Red gG2.
Movie S4. MPIs of MPM z stack images of ventral ear sections from LysM-eGFP mice infected with HSV-1 Red.
Movie S5. MPIs of MPM z stack images of ventral ear sections from LysM-eGFP mice infected with HSV-1 Red gG-SC.
Movie S6. MPIs of MPM z stack images of ventral ear sections from LysM-eGFP mice infected with HSV-1 Red gG2.
Movie S7. MPIs of MPM z stack images of ventral ear sections from T-bet-eGFP mice mock- infected.
Movie S8. MPIs of MPM z stack images of ventral ear sections from LysM-eGFP mice mock- infected.
Table S1. List of oligonucleotides used in this thesis.
Table S2.
List of mutations (SNPs and InDels) from variant analysis of sequenced HSV-1 original stock and isolated clones 1-5.
Table S3.
List of mutations (SNPs and InDels) from variant analysis of sequenced HSV-2 original stock and isolated clones 1-5.
Table S4.
List of mutations (SNPs and InDels) from variant analysis of deeper sequenced HSV- 1 isolated clones 2 and 3.
List of figures, tables and movies Table S5.
List of mutations (SNPs and InDels) from variant analysis of deeper sequenced HSV-
2 isolated clones 1 and 5.
Table S6.
List of mutations (SNPs and InDels) from variant analysis of HSV-1 clone 2 genomic variability study after 0 (P0), 5 (P5) and 10 (P10) passages in Vero cells.
Table S7.
List of mutations (SNPs and InDels) from variant analysis of HSV-1 clone 2 genomic variability study after 0 (P0), 5 (P5) and 10 (P10) passages in HaCaT cells.
Table S8.
List of mutations (SNPs and InDels) from variant analysis of HSV-1 clone 3 genomic variability study after 0 (P0), 5 (P5) and 10 (P10) passages in Vero cells.
Table S9.
List of mutations (SNPs and InDels) from variant analysis of HSV-1 clone 3 genomic variability study after 0 (P0), 5 (P5) and 10 (P10) passages in HaCaT cells.
Table S10.
List of mutations (SNPs and InDels) from variant analysis of HSV-2 clone 1 genomic variability study after 0 (P0), 5 (P5) and 10 (P10) passages in Vero cells.
Table S11.
List of mutations (SNPs and InDels) from variant analysis of HSV-2 clone 5 genomic variability study after 0 (P0), 5 (P5) and 10 (P10) passages in Vero cells.
Table S12.
List of mutations (SNPs and InDels) from variant analysis of HSV-2 clone 5 genomic variability study after 0 (P0), 5 (P5) and 10 (P10) passages in HaCaT cells.
Table S13.
List of mutations (SNPs and InDels) from variant analysis of sequenced HSV-1 strain 17 wt and BAC derived viruses.
Table S14.
List of mutations (SNPs and InDels) from variant analysis of sequenced HSV-2 strain MS wt and BAC derived viruses.
Table S15.
List of mutations (SNPs and InDels) from variant analysis of sequenced HSV-1 recombinant viruses, including Red, ∆gG-eGFP and gG mutant viruses.
Table S16.
List of mutations (SNPs and InDels) from variant analysis of sequenced HSV-2 recombinant viruses, including Red, ∆gG-eGFP and gG mutant viruses.
Table S17.
List of gRNAs, mapping with 0 mismatches, found in UL26-27 intergenic region of HSV-1 wt and HSV-2 wt genomes.
Table S18.
List of gRNAs, mapping with 0 mismatches, found in US4 locus of HSV-1 Red and HSV-2 Red genomes.
Table S19.
List of gRNAs, mapping with 0 mismatches, found in CMV enhancer and bGH polyA signal regions into eGFP cassette inserted in US4 locus of HSV-1 Red and HSV-2 Red genomes.
Table S20.
List of sequenced viral stocks and samples in this thesis.
1. Abstract / Resumen
1. Abstract / Resumen Herpes simplex virus (HSV) is well-known for being one of the most prevalent neurotropic pathogens worldwide, causing a broad range of diseases in humans. Two different HSV subtypes have been traditionally associated to diverse clinical manifestation of the disease, being HSV type 1 (HSV-1) global seroprevalence much higher than that of HSV type 2 (HSV-2). Whereas HSV-1 is generally the causative agent of cold sores, keratitis or even encephalitis by infecting the oro-facial region, HSV-2 tends to infect the genital area causing pain, heat and itch, which are classical symptoms of genital herpes. Despite being both able to infect the genitalia, HSV-1 recurrent episodes are less frequent than HSV-2 infections. These data suggest that clear differences exist when comparing HSV-1 and HSV-2 pathogenesis and prevalence, suggesting a differential neurotropism for each virus. How these highly related viruses differentially manipulate the host nervous system is not properly understood yet.
HSV glycoprotein G (gG) is the most divergent viral product between the HSV subtypes, which has been identified by our laboratory as the first viral proteins able to enhance chemotaxis both in vitro and in vivo. Both HSV-1 gG (gG1) and the secreted domain of HSV-2 gG (SgG2) showed chemotaxis potentiation. In addition, SgG2 was described as the first viral protein inducing a directional enhancement of nerve growth factor (NGF)-mediated axonal growth of neurons ex vivo and in vivo.
Our hypothesis is that these novel biological activities, together with the structural differences among gG1 and HSV-2 gG (gG2), may contribute to explain the preferential neurotropism of each virus.
Here, we have determined the genomic sequence of some relevant HSV laboratory strains, characterizing their genomic and genetic variability within each HSV subtype in cell culture, prior to further studies. Then, we have optimized the CRISPR/Cas9 system to efficiently generate a collection of gG mutant HSVs in order to investigate the contribution of both gG1 and gG2 to viral pathogenicity and neurotropism.
We observed that gG1 is dispensable for HSV-1 infection, while gG2 appears as a non-essential but important contributing factor in HSV-2 pathogenesis. Swapping of gGs between the HSV subtypes led to a dramatic HSV-1 attenuation, which was less pronounced in the HSV-2 case. Together with previous data, our results suggest that gG2 may facilitate access of HSV to NGF-dependent neurons, giving a different outcome of infection depending on which HSV subtype infects those neurons. We propose that gG2, likely SgG2, may play a crucial role during HSV-2 colonization of the nervous system, contributing to explain its differential neurotropism with HSV-1.
1. Abstract / Resumen El virus herpes simplex (HSV) es bien conocido por ser uno de los patógenos neurotrópicos más prevalente en todo el mundo, causando un amplio rango de enfermedades en humanos. Dos subtipos diferentes de HSV han sido tradicionalmente asociados a diversas manifestaciones clínicas de la enfermedad, donde la estimación de la seroprevalencia global de HSV tipo 1 (HSV- 1) es mucho mayor que la de HSV tipo 2 (HSV-2). Mientras que HSV-1 es generalmente el agente causante del herpes labial, queratitis o incluso encefalitis por infectar la zona oro-facial, HSV-2 suele infectar el área genital causando, dolor, calor y picazón, los cuales son síntoma clásicos del herpes genital. A pesar de que ambos son capaces de infectar los genitales, los episodios recurrentes por HSV-1 son menos frecuentes que en las infecciones por HSV-2. Estos datos sugieren que existen claras diferencias comparando la patogénesis y prevalencia de HSV-1 y HSV-2, sugiriendo un neurotropismo diferencial para cada virus. Aún no entendemos propiamente cómo estos virus altamente relacionados manipulan diferencialmente el sistema nervioso del hospedador.
La glicoproteína G de HSV (gG) es el producto viral más divergente entre ambos subtipos de HSV, la cual ha sido identificada por nuestro laboratorio como la primera proteína viral capaz de incrementar la quimiotaxis in vitro e in vivo. HSV-1 gG (gG1) y la porción secretada de HSV-2 gG (SgG2) mostraron potenciación de la quimiotaxis. Además, SgG2 fue descrita como la primera proteína viral que induce una potenciación del crecimiento axonal de neuronas mediado por el factor de crecimiento nervioso (NGF), ex vivo e in vivo. Nuestra hipótesis es que estas novedosas actividades biológicas, junto con las diferencias estructurales entre gG1 y HSV-2 gG (gG2), pueden contribuir a explicar el neurotropismo preferencial de cada virus.
En este trabajo hemos determinado la secuencia genómica de algunas cepas de laboratorio relevantes de HSV, caracterizando su variabilidad genómica y genética en cultivo celular dentro de cada subtipo de HSV, antes de realizar estudios posteriores. Hemos optimizado el sistema CRISPR/
Cas9 para generar eficientemente una colección de virus mutantes en gG, con el fin de investigar la contribución de gG1 y gG2 en la patogenicidad viral y el neurotropismo.
Observamos que gG1 no es necesaria para la infección de HSV-1, mientras que gG2 aparece como un factor no esencial pero importante, que contribuye a la patogénesis de HSV-2. El intercambio de las gGs entre los diferentes subtipos de HSV conduce a una dramática atenuación de HSV-1, que fue menos pronunciada en el caso de HSV-2. Junto a los datos previos, nuestros resultados sugieren que gG2 puede facilitar el acceso de HSV a neuronas NGF-dependientes, dando un resultado de la infección diferente dependiendo de qué subtipo de HSV infecte esas neuronas. Proponemos que gG2, probablemente SgG2, puede tener un papel crucial durante la colonización del sistema nervioso por HSV-2, contribuyendo a explicar su neurotropismo diferencial con HSV-1.
2. Introduction
2. Introduction 2.1. HUMAN ALPHAHERPESVIRUSES
Herpesviruses, together with Influenza and Variola viruses, are some of the best known viral human pathogens worldwide. Early in human history, physicians in the ancient Greek recognized disease symptoms describing cutaneous lesions that “snaked” over patient’s skin (herpein in Greek).
Since the discovery of human herpesviruses, extensive research has been generated in both basic and clinical research linking their infection to human diseases. However, despite considerable effort, the strategies developed for efficient treatment and prevention, such as anti-viral drugs and vaccines, are very limited.
To date, Herpesviridae comprehends a large family of viruses composed by more than 130 members with unique biological features [1, 2]. Within them, eight human herpesvirus species are known, and each can cause a variety of diseases exhibiting a broad range of symptoms and severities [3]. The defining characteristic of the Alphaherpesvirinae subfamily is the ability to establish life-long latent infection in sensory ganglia of their host [4, 5]. In humans, alphaherpesviruses encompass varicella zoster virus (VZV) and two of the most prevalent and ubiquitous human pathogens, HSV-1 and HSV-2.
HSV-1 infects most commonly the oral mucosa and less frequency the genital mucosa, causing characteristic minor lesions or cold sores. Genital herpes is more often caused by HSV- 2. Nevertheless, both viruses can infect either mucosa, frequently as a consequence of oral- genital sex [6-8]. In addition to these common symptoms, HSV can also cause lesions in fingers (herpetic whitlow), at sites of abrasion (herpes gladiatorum), and in the eyelids (herpes blepharitis), degenerating in conjunctivitis. Genital herpes during late pregnancy can be very damaging for neonates [9]. Moreover, infection of the corneal tissue in the eye leads to herpes keratitis but the most serious disease occurs when the virus penetrates the central nervous system (CNS), causing herpes encephalitis. Although HSV infections are usually limiting in immunocompetent individuals, morbidity and mortality increase in immunocompromised patients [10].
The latest epidemiological studies estimate around 67% and 11.3% of global seroprevalence for HSV-1 and HSV-2, respectively, depending on the geographical region [11, 12]. Approximately 14,000 newborns are infected with HSV each year globally [13]. Around 300,000 cases of ocular herpes are diagnosed annually only in USA, where also there is an estimation of 1,500 cases of herpes encephalitis with considerable mortality, despite the accessibility of antiviral drugs. Moreover, genital herpes infection increases the probability of HIV infection and transmission by 2-4 fold [14].
Thus, since antiviral approaches only fight against the lytic infection, reservoirs of HSV remain latent in human neurons. New therapeutic strategies and further research to identify new strategies of eradication and prevention are a public health priority worldwide.
2.2. HSV BIOLOGY
2.2.1. Life cycle
The life cycle of HSV involves both lytic and latent stages, which are characterized by productive and non-productive infections, respectively [15]. An initial replication upon entry at the broken skin or mucosal surface leads to a productive infection, allowing viral dissemination to other hosts and more importantly, access to free nerve endings (FNEs) of the surrounding sensory neurons which innervate the tissue [16, 17]. Viral particles enter the neuronal axons and nucleocapsid and tegument components travel by retrograde transport to the neuronal soma, where the viral genome is released into the nucleus (Figure I1a, upper chart). This is an active transport process in which viral and host components participate [18, 19]. Viral DNA persists in the nucleus in a circular episomal form that is associated with nucleosomes [16]. Once the virus reaches the trigeminal ganglia (TG) or the dorsal root ganglia (DRG), as it is usually the HSV-1 and HSV-2 cases, respectively, the virus establishes latency for the lifetime of the host. Lytic gene expression is repressed, but the latency- associated transcript (LAT), a non-coding RNA (ncRNA), is highly expressed in some (around 30%), but not all, latently infected neurons [20-22], accompanied by several micro RNAs (miRNAs) [23-25].
LAT and some miRNA expression help to silence lytic infection promoting the maintenance of the latent state. Although practically all the infected neurons have a severe lytic gene repression, some studies have shown low levels of active viral production, proponing HSV infection as a persistent rather than a latent infection [26-28]. In humans, spontaneous and induced reactivation (e.g., fever, UV light, psychological stress [29]) involve expression of early and late proteins and viral DNA replication, components of the new virions which reach the axonal tip by anterograde transport to neuronal branches [16, 30], where new virions are assembled and released into the mucosal periphery (Figure I1a, lower chart). Here, recurrent infection and transmission occur, exhibiting associated disease symptoms.
2.2.2. Lytic stage
The entry of viral particles into cells is a complex multi-step process which involves cellular receptors on the host cell surface and viral proteins in the viral envelope [31]. Virus attachment to the cell surface is mediated by viral glycoproteins B (gB) and C (gC) with cellular glycosaminoglycans (GAGs), facilitating glycoprotein D (gD) interaction with cellular receptors. Viral envelope fusion with the cell plasma membrane is triggered by gD and gB, recruiting the required heterodimeric glycoproteins H and L complex [32]. Some molecules have been identified as HSV entry receptors, like herpes virus entry mediator (HVEM), nectin-1/-2 and 3-O heparan sulfate [33-35], contributing in part to the broad HSV cell tropism [36]. In addition, GAG interactions are not essential despite enhancing viral infectivity. Viral entry can occur predominantly by fusion with the cell membrane (pH-
2. Introduction
Figure I1. Life cycle stages of HSV. (a) Initial infection of the epithelial mucosa results in a first productive replication, where infection can spread locally. Viral particles reach surrounded free nerve endings (FNE) of sensory neurons and nucleocapsid travel by retrograde transport to the neuronal soma, establishing latency in the nucleus as episomal DNA. Reactivation triggers lytic gene expression, producing viral components which are anterograde transported until axonal termini. Infectious progeny is released, infecting epithelial cells and causing tissue damage and disease symptoms. (b) Lytic cycle flowchart. Naked nucleocapsid reaches the nuclear pore to deliver viral genome into the cell nucleus. 1: Immediate-early (IE) gene are transcribed by host RNA polymerase II. 2: IE genes promote early (E) gene expression, which includes proteins requires for genome replication. 3: Viral DNA replication triggers late (L) gene expression, leading to the production of viral structural proteins. 4, 5: Capsid assembly and DNA encapsidation occur in the nucleus. 6-8: viral progeny maturation and egress by exocytosis. Modified from [16].
independent) or through endocytosis (pH-dependent) [37]. It has been proposed that the viral entry route may depend on the cell type and polarization, where gG could play a role infecting the apical surface of polarized cells [38].
The naked viral capsid is transported along microtubules to the nuclear membrane, where it associates with nuclear pores to release the viral genome, entering the nucleoplasm. The gene expression cascade is heavily regulated (Figure I1b), where viral and cellular proteins participate [1, 4, 15, 16]. Immediate-early genes are firstly transcribed using the cellular machinery, stimulated by the viral tegument protein VP16. Early genes are transcribed once immediate-early proteins have been synthesized. Independently, viral DNA replicates and, after that, late genes are transcribed.
Following capsid assembly and DNA packaging in the nucleus, viral particles bud through the outer nuclear membrane by fusion, losing their envelope and releasing nucleocapsids into the cytosol.
Capsids are re-enveloped acquiring mature glycoproteins when budding into Golgi apparatus vesicles. Mature viral progeny is released by exocytosis by fusion of the vesicle with the plasma
Replication compartment
Nucleus
IE mRNA
E mRNA
E proteins IE proteins
L proteins
L mRNA
1 2
3
4
5 6
7
8
(a) (b)
membrane. Cell-to-cell spread contributes to viral propagation, which is mediated by glycoproteins E (gE) and I (gI), serving as a key mechanism in lateral spread and immune evasion [39].
2.2.3. Virion and genome structure
The diameter of the mature HSV particle is 180-200 nm and consists, as shown in Figure I2a, of a lipid envelope, an icosahedral capsid organized in 162 capsomers and a double-stranded DNA (dsDNA) genome [40, 41]. Between the capsid and the envelope there is an amorphous layer known as tegument, containing more than 30 viral proteins. The lipid envelope contains at least 11 viral glycoproteins, which are involved in diverse aspects of the viral life cycle. These glycoproteins trigger potent immune responses, being key targets for immunization strategies [42, 43]. Curiously, all viral glycoproteins elicit cross-reactive T and B cell responses, but not gG, which induces HSV type specific antibody responses allowing its clinical used as a serological indicator to differentiate between HSV-1 and HSV-2 infections [44]. The lack of cross-reactivity between gG1 and gG2 has been related to significant differences at the structural level [45].
The HSV genome has 152-155 Kilobase pairs (Kbp), varying slightly between laboratory strains and clinical isolates. Nearly 80 genes have been identified, including proteins for initial gene expression, shut off of host cell, maturation and egress and immunomodulation [2, 4, 15]. The HSV genome is organized in a number of distinct segments, comprising two major unique (U) regions, termed long (UL) and short (US) regions, flanked by large internal (I) or terminal (T) inverted repeats, i.e., TRL, IRL, IRS and TRS (Figure I2b). The “a” sequence is present at the IRL-IRS border, but also at the termini of TRL and TRS; enabling the inversion of the unique fragments orientation and producing four genomic isomers in equal ratios and functionality [46, 47]. The HSV-1 and HSV-2 genomes are highly conserved, but the US region is more divergent between them (Figure I2c).
Here, the US4 gene, which encodes gG, appears as the most divergent gene between those viruses [48, 49], and represents the main focus of this thesis.
2.2.4. Serotype-dependent neurotropism
HSV epithelial replication is accompanied by infection of FNE and subsequent retrograde axonal transport to the soma of sensory neurons, where infection may trigger a productive or a latent stage. At this point, some neurons die after new progeny production, whereas in other cases the lytic phase is repressed and the virus establishes latency. Different factors may determine which phase of the viral cycle predominates. Despite those factors remaining unclear, there is evidence supporting that different neuronal subpopulations or some host factor can be critical to tilt the balance towards one or other outcome.
As mentioned above, HSV-1 is most commonly found establishing latency in TG whereas, in contrast, HSV-2 tends to become latent in DRG. Both are sensory ganglia, clustering neurons
2. Introduction
Figure I2. HSV virion and genomic structure. (a, left) Electron microcopy image of an HSV virion particle, indicating structural elements. Adapted from [15]. (a, right) Cutaway schematic view of virion structure (T=16, icosahedral symmetry). Credit: ViralZone 2017, Swiss Institute of Bioinformatics. (b) HSV genome (prototype orientation). The genome segments and repeated sequences are identified as described in the text. CDSs are presented in forward (red) or reverse (blue) orientation. (c) Nucleotide sequence similarity between HSV-1 and HSV-2. US segment is magnified on the right side, showing the greatest divergence in the US4 gene. Modified from [48].
(a)
(b)
(c)
IRS TRSUS
RS1 RS1
US10-12 US3 US6
US4
Similarity Score
10
5
0 150,000
implicated in a variety of signaling events to the CNS, some of which included temperature, pressure, and pain. Thus, sensory neurons have been classified accordingly to different parameters such as myelination, expression of neuropeptides and neurotrophic dependency, among others [50]. Margolis and colleagues described that the A5 and KH10 markers, which recognize different uncharacterized profiles of carbohydrates, identify distinct nociceptive neuronal populations in mouse sensory ganglia
[51]. The majority of A5+ neuronal population also immunoreacts for calcitonin gene related peptide (CGRP) and the tropomyosin receptor kinase A (TrkA) [52], the latter being a receptor for NGF. On the other hand, KH10+ neurons are additionally IB4+ (vegetal isolectin B4) and express the receptor for glial cell-derived neurotrophic factor (GDNF) [53, 54]. Margolis’s team showed that productive infection after viral entry in neurons from adult mouse cultured TG was similarly restricted approx.
up to 55% for HSV-1 and HSV-2 infections [54]. However, they and others interestingly found that HSV-1 establishes latency and expresses LAT more often in A5+ TG and DRG neurons, while HSV- 2 latency is more common in the KH10+ population [52, 55, 56]. Conversely, HSV-1 and HSV-2 are able to replicate more productively in KH10+ and A5+ cultured TG neurons, respectively [54, 57]. This correlates with the symptoms associated with genital herpes, such as pain, heat and itch, since the A5+ population are peptidergic nociceptive neurons [58, 59]. Moreover, NGF signaling has been reported as a requirement to maintain in vitro HSV-1 latency, whereas GDNF ligand-receptor is a relevant factor in the HSV-2 silent stage [60, 61]. Hence, it seems that host neurotrophic factors and their neuronal receptors may contribute to the differential HSV serotype neurotropism and to the lytic/silent outcome after infection [62].
2.3. GENOMIC AND VIRULENCE VARIABILITY
2.3.1. Replication and recombination of the viral genome
Viral DNA replication begins in the cell nucleus after early gene expression has commenced.
HSV has three origins of DNA replication, one within UL the segment (oriL) and two (oriS) in the IRS and TRS repeated regions, respectively. Different functions for the two types of origins during lytic and latent infections have been proposed. While mutant viruses containing point mutations in the oriS behaved in vivo like wt viruses, those with similar mutation in the oriL reduced their virulence [63], suggesting that oriL plays a role in in vivo replication and pathogenesis.
The viral genome encodes all required proteins for DNA self-replication, including the origin recognition protein (UL9), a helicase/primase complex (UL5, UL8 and UL52), a ICP8 DNA-binding protein (UL29) and the viral DNA polymerase (UL30 and UL42) [15]. Proteins involved in nucleotide metabolism are also expressed, spotlighting UL39, UL40, UL50, UL2 and UL23, the latter encoding a thymidine kinase, which is the target of the most commonly used anti-HSV drug (acycloguanosine, a.k.a. acyclovir). The DNA polymerase is composed of two subunits: catalytic (UL30) and processivity (UL42) subunits, both essential for viral replication. UL30 exhibits 3’-5’ exonuclease activity and apurinic/apyrimidic lyase activities, where mutations in its CDS may reduce replication fidelity [64- 66]. In addition, mutations in UL42, which reduce DNA binding, are also related with reduction in viral DNA replication fidelity [67].
2. Introduction DNA replication produces long concatemers of viral DNA which are processed into unit length genomes after cellular endonuclease G cleavage in the “a” sequence [68]. The alkaline exonuclease UL12 is required for processing of those unit length DNA molecules. Those concatemers are highly branched, promoting recombination events between repeated regions and resulting in the inversion of the UL and US segments [69]. Interestingly, high rates of recombination and inversion in the HSV genome have been described in the human populations [70, 71], which suggests that those are key components in generation of viral diversity and evolution.
2.3.2. Generation of genomic/genetic variability
Traditionally, DNA viruses have been classified as more stable than RNA viruses, since most DNA viruses have high fidelity (HF) polymerases with error correction, i.e. proofreading mechanisms.
Although early studies proposed a mutation rates of 10-7-10-8 for herpesviruses such as HSV-1 [72, 73], these data do not correlated with the rapid ability observed when selecting HSV variants under drug pressures [74]. In fact, clinical isolates from patients not treated with antiviral drugs showed preexisting subpopulation of acyclovir resistant virus [75]. On the other hand, it has been reported that the HSV-1 DNA polymerase misincorporates dNTPs with a frequency of approx. 3 x 10-3 [76, 77], which is nearly 10-fold higher than the frequency reported for the exonuclease-deficient Klenow fragment of E. coli DNA polymerase I [78]. Mutations in genome replication occur frequently during HSV infections, where different mutations, such as single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels), can be detected across viral genomes [79, 80]. Thus, the variability generation rate in HSV viral progenies may be higher than previously predicted.
HSV has evolved a complex relationship with the host DNA damage response pathways [2]. Numerous cellular factors involved in double-strand break (DSB) repair are recruited to viral replicative compartments. Recruitment of those host factors suggests that one or more DSB repair pathways may be activated during HSV infection as previously described [81]. It was found that single-strand annealing is increased in HSV infections by UL12 [82]. Single-strand annealing is a high error prone form of homology-mediated repair cellular mechanism, which can cause deletions and translocations. These data support the notion that the HSV recombination process is intrinsic to replication, contributing to evolution in herpesviruses through genome repair, and generation of isomers and variability.
Cell culture techniques are essential for HSV studies and development of antiviral drugs and vaccines. Nonetheless, it has been reported that high number of cell culture passages and amplification can induce undesirable selective pressure, as notably described in human cytomegalovirus (CMV), a member of the betaherpesvirus subfamily [83, 84]. Frequent deletion of the UL55-56 genomic region has been detected in some HSV-1 strains [79]. Improvements in DNA sequencing, as well as the low cost and accessibility, have been pressing to characterize genomic changes by next generation
Table I1. HSV strains commonly used in experimental research. Modified from [88]. HSV-1 SC16 was manually included from [221].
sequencing (NGS) technologies during HSV propagation in cell culture or after any modification introduced during the generation of mutant viruses.
2.3.3. Viral isolates, strains and consensus genomes
The HSV serotype and specific viral strain determine the preferred cell type for infection and the ability to colonize the CNS and to evade the host immune system. There is a variety of clinical isolates and laboratory-adapted strains which can diverge significantly in the severity of the disease, even within the same subtype (Table I1). Mutational analysis has revealed viral determinants of virulence affecting strain pathogenesis severity and neurovirulence [85-88].
Conventionally, a virus collected from a clinical source is called an isolate, which can be referred to as a strain after cell culture amplification. However, the viral population can change through random genetic drift after subsequent rounds of amplification or during intentional bottlenecks such as plaque isolation when generating mutant viruses (Figure I3). Therefore, a viral strain can easily become a mixed population of viruses, whose variants could evidence differences in observable phenotypes. A well-studied case is the HSV-1 strain KOS, since it has generated variants by genetic drift over passages in cell culture and plaque purification [85, 89]. These variants exhibited a broad spectrum in their ability to induce immune responses [90], encephalitis severity in animal models of pathogenesis [88, 91], and expression of viral antigens [92].
Those previous experiments were performed using a heterogeneous population of genome- containing virions. NGS technologies have revealed the question of viral population diversity and their implications. Usually, herpesvirus diversity has focused on defining the consensus genome of preexisting strains and isolates, which represents the most common nucleotide at each position across the genome (Figure I3a). Although the consensus genome is derived from the most common sequenced variant of the population, it may significantly vary over time regarding to the original isolate
Strain name Source Relative virulence in adult mice
HSV-1 McKrae Keratitis isolate +++
HSV-1 KOS Recurrent oral lesion +
HSV-1 RE Keratitis isolate ++
HSV-1 17 syn+ Not disclosed +++
HSV-1 F Facial or genital lesion +
HSV-1 Patton Recurrent oral lesion +
HSV-1 SC16 Oral lesion +++
HSV-2 186 Penile lesion +++
HSV-2 333 Primary genital lesion +++
HSV-2 MS Midbrain of patient with multiple sclerosis ++
2. Introduction
due to the factors mentioned above (Figure I3b). Even superficial sequencing could eventually give a consensus genome sequence which would not be representing the most common variants at certain genomic locations. Recent studies have showed that deep coverage of large DNA viral genomes by short-read technologies have allowed the detection of minority variants with low frequency in the viral population, whereas long-read sequencing data have contributed to resolve the large and complex repeated regions in HSV genome [93-95].
2.4. TOOLS AND MODELS FOR HSV STUDIES
2.4.1. Systems to generate recombinant HSVs
The original method for generating recombinant viruses was based on spontaneous homologous recombination between a transient-transfected donor plasmid, containing the desired mutated sequence flanked by homology arms, and the viral genome, this latter provided to cultured cells by co-transfection or infection [96-98]. These spontaneous homologous recombination events occur with a very low rate, which means that a high number of viral plaques had to be screened to find a few recombinant viruses produced. Even using approaches such as the use of antibiotic, several rounds of selection were needed after plaque screening, making the process time-consuming and labor-intensive.
Figure I3. Viral genome populations contain a broad range of variations which may change their frequencies over time. (a) The consensus genome generated for a viral isolate reflects the most abundant sequence present in the viral population. The consensus sequence may not be represented in the majority of the individual genomes that may incorporate specific mutations in some positions. (b) Selective pressures can result in a minor variant becoming the most frequent in the population. Recombination and gene accordions may also generate new genotypes under restrictive conditions. Adapted from [233].
(a) (b)
More recently, bacterial artificial chromosomes (BACs) have been used as a promising system to enable the introduction of any desired mutation using tools of bacterial genetics. Briefly, a BAC origin of replication is firstly introduced into the HSV genome by homologous recombination in eukaryotic host cells. The HSV genome circularizes and this transient form is used to transform bacteria, and then, the cloned BAC can be used to edit the viral genome. After transfection of eukaryotic cells, modified viral progeny carrying the desired mutation is recovered [99]. However, this viral progeny usually contains residual BAC sequences and other unwanted changes [96, 100- 103]. Some of the most frequently characterized cases are the unintended alteration of viral origins of replication (oriL and oriS) and repeated regions, due to their repetitive structure. The palindromic sequence of the oriL is lost after BAC cloning and is not maintained in any HSV BAC [99, 103, 104].
Although it has been suggested that the oriL deletion has no impact in cell culture, its influence on the acute infection and reactivation from latency in animal models remains poorly characterized, since only one previous study indirectly suggested that it might impair in vivo virulence and pathogenesis [63]. In addition, “a” sequences are susceptible to shorten after bacterial passage [102, 103], which could lead to the loss of the DNA packaging signal sequence contained into the “a” sequence [47, 105].
Site-specific genome editing tools have been broadly used to target genes within any organism. In the past few years, Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated protein 9 (Cas9) have revolutionized genome engineering across laboratories worldwide [106, 107]. CRISPR/Cas9 system is part of the bacterial acquired immune system to cleave invasive DNA (Figure I4a). Nuclease Cas9 can use a synthetic guide RNA (gRNA) to direct DNA cleavage. In contrast to other genome editing methods that require protein engineering for each DNA target site to be modified, the CRISPR/Cas9 system only requires a change in the guide RNA sequence to modify target specificity. The target recognition has two requirements: base pairing to the gRNA 20-nucleotide at the 5’ end and the presence of a protospacer adjacent motif to the target sequence (Figure I4b). However, despite exhibiting high specificity, Cas9 can tolerate up to five mismatches between the gRNA and target DNA, in a sequence-dependent manner, promoting undesired off-target mutations [108, 109]. Due to that, numerous strategies have been implemented to improve the specificity of Cas9-mediated genome editing [110, 111]. Cas9 generates DSB at the target site, which triggers cellular DNA repair mechanisms, through error-prone non-homologous end joining (NHEJ) to introduce gene disruptions, and homology-directed repair (HDR) by the insertion of donor sequences (Figure I4c).
Some studies have reported a successful genome editing in herpesviruses, proving high specificity to generate mutant HSVs by both NHEJ and HDR [112-114]. Together with others studies in the field, HSV research in basic biology, pathogen treatment and gene therapy have improved [115]. For instance, CRISPR/Cas9 technology was utilized not only to introduce point mutations or to
2. Introduction
Figure I4. Schematic of the CRISPR/Cas9 system involved in bacterial acquired immunity and their application in genome editing procedures. (a) Bacterial adaptive immunity occurs in three different stages:
(1st) acquisition of a short sequence of invading DNA (the protospacer) and its insertion into the CRISPR array as a spacer, involving cleavage of the protospacer by Cas proteins; (2nd) transcription of the pre-crRNA to generate later mature crRNAs; and (3rd) crRNA-directed cleavage of foreign DNA by Cas proteins at sites complementary to the crRNA spacer sequence, known as interference. There are three system types, using distinct molecular mechanisms for interference. (b) Structure and function of the Cas9-sgRNA complex.
Cas9 uses tracrRNA and crRNA to direct DNA cleavage, which can be engineered as a synthetic gRNA (sgRNA). Target recognition requires both base pairing to the 20-nucleotides sequence at the 5’ end of sgRNA and the presence of a protospacer motif (PAM) that is adjacent to the targeted sequence. (c) DSBs induced by Cas9 can be repaired in one of two directions in mammalian cells. By the error-prone NHEJ pathway, the ends of a DSB are processed by the endonegous DNA repair machinery and rejoined, resulting in random indel mutations. Otherwise, a repair template can be provided to activate the HDR pathway, allowing high fidelity and precise editing. Figures (a) and (b) were adapted from http://www.nature.com/
nrmicro/posters/crispr/index.html; and scheme (c) from [108].
replace viral genes by the enhanced green fluorescent protein (eGFP), but also to efficiently inhibit HSV replication during acute infection and latency [116-118]. However, HDR efficiency rates are not well characterized in many cases, neither the selection steps to increase the frequency of DSB events nor to promote HDR. Altogether, these data propose CRISPR/Cas9 technology as a powerful tool to generate recombinant viruses, although improvements are still needed.
2.4.2. Murine models of HSV pathogenesis and latency
Multiple animal models have been used to investigate HSV pathogenesis and the antiviral immune response, where mouse is the most widely species utilized [88]. HSV mouse infection shows a diffuse pattern of viral spread and lesions in the mouse brain, where the virus is able to
Acquisition Spacer
New spacer
pre-crRNA crRNA maturation RNase
Repeat duplication
Type I systems
Cascade complex
Invading DNA
Cas9 complex Csm or Cmr complex crRNA
Invading DNA
crRNA crRNA
5ʹ 3ʹ
Type II systems tracrRNA
Type III systems CRISPR locus Leader
Protospacer dsDNA
PAM
cas genes
1
crRNA biogenesis 2
Interference 3 3ʹ
5ʹ 3ʹ 5ʹ
Invading DNA Invading RNA
Repeat
-
NHEJ HDR
DSB
5ʹ3ʹ 3ʹ
5ʹ sgRNA Cas9
|||
|||
Indel mutation Premature
codonstop Precise gene editing
Genomic 5ʹ DNA 3ʹ 5ʹ3ʹ
5ʹ3ʹ
3ʹ5ʹ
3ʹ5ʹ
Repair 5ʹ template 3ʹ
5ʹ3ʹ
3ʹ5ʹ 3ʹ5ʹ
3ʹ5ʹ
(a)
(c) (b)
Cas9 sgRNA
Genomic DNA
Matching genomic sequence
PAM
Animal cells Bacteria and parasites
Human cells
Repair
Targeted genome editing
Donor DNA
rapidly stablish latency at the neuronal ganglia within the first 24 hours post-infection (hpi), although reactivation do not appear to occur spontaneously [119]. Mice support productive viral replication and have been genetically manipulated to generate an extensive variety of modified strains. In addition, the availability of research reagents in mice has accelerated the complex analysis of immunological and antiviral responses against infection. Overall, this provides a powerful model for studying diverse aspects of the initial infection, innate and adaptive immune responses, neuroinvasion, antiviral drugs and vaccines testing, as well as establishment, maintenance and reactivation from latency.
Mouse strain, age and route of inoculation determine the spectrum of resistance to HSV infection. Inbred strains of mice show a spectrum of susceptibility to HSV infection, CNS colonization and pathogenesis [120]. C57BL/6 mice display the highest levels of resistance (LD50 > 106 plaque- forming units (PFUs)) and restricted access of HSV across the CNS; the BALB/c strain showed intermediate susceptibility with elevated viral spread in the brain; and AKR and SWR mice exhibited the lowest resistance (LD50 ~ 10 PFU) [121, 122]. The restricted viral spread from the inoculation site to the CNS correlates with the protection against the disease severity, despite the high frequency of latency establishment at the TG being similar between all murine strains [122]. On the other hand, immune responses are strongly different when compared newborn to adult mice, where animal models of HSV infection have shown higher vulnerability in younger animals. For instance, HSV-1 infection in neonatal mice have proven lower early type I interferon (IFN) production and, therefore, increased viral replication than that observed in older animals [123, 124].
Depending on the HSV life cycle and the different experimental parameters to address, multiple inoculation sites have been used to study diverse viral and host events (Table I2). Delving into some of them, intranasal (i.n.) inoculations have been used to induce encephalitis, to establish latency and to test antiviral drugs; since HSV invades both olfactory and trigeminal nerves, spreading to the CNS [125, 126]. Intravaginal (i.v.) infections closely mimic human genital HSV disease. Female mice are pretreated with medroxyprogesterone acetate prior to infection, to synchronize estrous cycles and promote viral uptake. HSV spreads through the genitourinary apparatus, accessing the sacral root ganglia to establish latency. The virus can also spread via the sacral root ganglia to the autonomic ganglia of the enteric nervous system in the colon [127]. This model is usually chosen to study innate immune responses, vaccine testing and spontaneous reactivation (this latter only in guinea pigs) [119, 128]. Ocular infections are also widely implemented to investigate latency establishment, spontaneous and induced reactivation, viral colonization of the CNS and activation-migration of T lymphocytes [22, 129, 130].
2. Introduction
Table I2. Different routes of inoculation for experimental studies of HSV animal models. Adapted from [88].
Anatomical location
of inoculation Commonly studied experimental parameters Species used Antigen uptake by dendritic cells and their migration to draining lymph nodes
T cell activation kinetics and migration T cell activation kinetics and migration
Mechanisms governing establishment and maintenance of latency Host strain susceptibility and HSV spread to CNS
Role of various components of innate immunity in controlling acute infection Host strain susceptibility and HSV spread to CNS
Role of specific inflammatory genes in limiting virus spread Migration of inflammatory cells to CNS
Involvement of neutrophils and T cells in severity of herpetic stromal keratitis Mechanisms governing establishment and maintenance of latency
T cell activation kinetics and migration
Role of various components of innate immunity in controlling acute infection Mechanisms of recurrent disease
Vaccine efficacy
Role of HSV genes in promoting viral replication and neurotoxicity Role of CNS-resident cells in controlling/promoting encephalitis Corneal epithelium
Intravaginal Intracranial
Mice Mice Mice Mice Mice, rabbits
Mice, guinea pigs Mice Flank skin
Rear footpads Oral mucosa Nasal mucosa
2.5. HOST IMMUNE RESPONSE AND HSV EVASION
The key feature of human herpesviruses is that these viruses have achieved an equilibrium with their host, as a result from their ability to establish a lifespan latent infection in neurons. To accomplish this goal, herpesviruses have dedicated the majority of their genomic coding capacity to express an enormous array of genes to evade and modulate the host immune responses. The study of this wide variety of immune evasion mechanisms has provided insights into fundamental aspects of both HSV infection and the host immune system from the viral point of view [131].
2.5.1. Countermeasures against innate and adaptive immunity
The production of type I IFN represents the first line of the host defense, determining the pathogenesis of HSV infection [132, 133]. IFNs engage with the cell surface receptors and trigger signal transduction cascades which activate the synthesis of numerous IFN-stimulated genes (ISGs) establishing a cellular antiviral state. Toll-like receptors (TLRs) have been shown to play a relevant role recognizing HSV nucleic acid and protein, which ultimately results in the expression of cytokines with antiviral activities, particularly type I IFN [134]. HSV encodes proteins that counteract the IFN pathway. For instance, the viral protein ICP0 is required to inhibit the IFN regulatory factor 3, which mediates IFN and ISG induction [135]; and this viral protein also targets IFN production through the DNA sensor IFI16 [136]. The virion host shutoff factor, encoded by UL41, inhibits ISG expression by promoting the degradation of cellular mRNAs [137]. The viral protein ICP34.5 inhibits one of the major ISGs, the protein kinase R and prevents its mediated translation inhibition [138]. The tegument protein encoded by US11 inhibits oligoadenylate synthetase, another major ISG [139]. Additionally,