In Vitro Evolution of Herpes Simplex Virus 1 (HSV-1) Reveals Selection
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bioRxiv preprint doi: https://doi.org/10.1101/760629; this version posted September 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Kuny et al., manuscript for review August 2019 1 In vitro evolution of herpes simplex virus 1 (HSV-1) reveals selection 2 for syncytia and other minor variants in Vero cell culture 3 4 Chad V. Kuny1, Christopher D. Bowen1, Daniel W. Renner1, Christine M. Johnston2, Moriah L. 5 Szpara1# 6 7 1Department of Biochemistry and Molecular Biology, The Huck Institutes of the Life Sciences, 8 and Center for Infectious Disease Dynamics, Pennsylvania State University, University Park, PA 9 2Department of Medicine, University of Washington, Seattle, Washington, Vaccine and 10 Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 11 12 Short Title: In Vitro Evolution of HSV-1 13 14 #Corresponding Author: Moriah L. Szpara, [email protected] 15 16 Keywords: HSV-1, evolution, minor variant, viral fitness, syncytia 17 18 p.1 bioRxiv preprint doi: https://doi.org/10.1101/760629; this version posted September 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Kuny et al., manuscript for review August 2019 19 Abstract 20 The large dsDNA virus HSV-1 is often considered to be genetically stable, however it is known 21 to rapidly evolve in response to strong selective pressures such as antiviral drug treatment. Deep 22 sequencing analysis has revealed that clinical and laboratory isolates of this virus exist as 23 populations that contain a mixture of minor alleles or variants, similar to many RNA viruses. 24 Classical virology methods often used plaque-purified virus populations to demonstrate 25 consistent genetic inheritance of viral traits. Plaque purification represents a severe genetic 26 bottleneck which may or may not be representative of natural transmission of HSV-1. Since 27 HSV-1 has a low error rate polymerase but exhibits substantial genetic diversity, the virus likely 28 uses other mechanisms to generate genetic diversity, including recombination, contraction and 29 expansion of tandem repeats, and imprecise DNA repair mechanisms. We sought to study the 30 evolution of HSV-1 in vitro, to examine the impact of this genetic diversity in evolution, in the 31 setting of standard laboratory conditions for viral cell culture, and in the absence of strong 32 selective pressures. We found that a mixed population of HSV-1 was more able to evolve and 33 adapt in culture than a plaque-purified population, though this adaptation generally occurred in a 34 minority of the viral population. We found that certain genetic variants appeared to be positively 35 selected for rapid growth and spread in Vero cell culture, a phenotype which was also observed 36 in clinical samples during their first passages in culture. In the case of a minor variant that 37 induces a visually observable syncytial phenotype, we found that changes in minor variant 38 frequency can have a large effect on the overall phenotype of a viral population. p.2 bioRxiv preprint doi: https://doi.org/10.1101/760629; this version posted September 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Kuny et al., manuscript for review August 2019 39 Author Summary 40 Herpes simplex virus type 1 (HSV-1) is a common virus, affecting over half of the adult human 41 population, although it presents variable levels of disease burden and frequency of symptomatic 42 recurrence. Antiviral treatments for HSV-1 infections are available, but thus far attempts at 43 vaccine development have been foiled by insufficient immunity and/or viral escape. As a virus 44 with a double-stranded DNA genome, HSV-1 is generally considered to be genetically stable and 45 to have limited evolutionary potential. As these two statements are in conflict, we examined the 46 ability of HSV-1 to evolve in a standardized cell culture setting. We utilized two HSV-1 isolates 47 in this experiment, one with multiple viral genotypes present, which is similar to the viral 48 populations seen in clinical settings, and one with a highly clonal viral population, which is 49 similar to those often used in laboratory settings. After multiple rounds of replication, we 50 analyzed the sequences of each passaged population. We found that the mixed viral population 51 changed substantially over passage, and we were able to track specific genetic variants to 52 phenotypic traits. By comparison, evolution in the clonal virus population was more limited. 53 These data indicate that HSV-1 is capable of evolving rapidly, and that this evolution is 54 facilitated by diversity in the viral population. 55 56 Introduction 57 HSV-1 is a widespread pathogen in the alphaherpesvirus family [1]. It is prevalent across 58 the world, infecting an estimated 3.7 billion people, who experience a spectrum of disease 59 outcomes from asymptomatic infections to recurring skin lesions or rarely, encephalitis [2]. 60 HSV-1 productively replicates in epithelial cells and then latently infects neuronal cells, leading p.3 bioRxiv preprint doi: https://doi.org/10.1101/760629; this version posted September 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Kuny et al., manuscript for review August 2019 61 to lifelong persistence in the host. The virus can periodically reactivate from latency to 62 productively replicate in epithelial cells, with the potential for multiple cycles of latency and 63 reactivation from the neuronal reservoir over the life of an infected individual [1]. This 64 replication strategy has great advantages for viral persistence, and it also provides multiple 65 opportunities for the virus to evolve within a host. During each active replication event, the virus 66 may undergo selection in response to the cellular and immunological selective pressures that it 67 faces [3–7]. Following productive replication, the progeny viruses can either be transmitted to a 68 new host or may re-enter the nervous system of the current host, potentially expanding the latent 69 reservoir of virus for future reactivation cycles [8,9]. As the selective pressures between 70 epithelial and neuronal cellular environments are likely different, each cycle of latency and 71 reactivation may create new genetic diversity in the viral population, which may be followed by 72 a bottleneck upon the conversion to latency. 73 While cycles of latency and reactivation are a well-established and accepted aspect of 74 herpesvirus biology [1,10], the concept of HSV-1 exhibiting substantial genetic diversity and 75 evolutionary potential within an infected individual has only recently been established [6,11–15]. 76 As with other herpesviruses, HSV-1 has a large double-stranded DNA (dsDNA) genome, and 77 these viruses have generally been considered to be genetically stable in comparison to viruses 78 with an RNA genome [5,16]. However an increasing body of evidence supports the idea that 79 herpesviruses, including HSV-1, exist as a diverse genetic population in vivo [17–20]. A similar 80 level of diversity may exist in vitro as well, depending on the method of preparation (reviewed in 81 [14]). This genetic diversity can be generated through multiple mechanisms, including 82 polymerase error, copy number variation, and recombination [3,4,21]. The HSV-1 polymerase 83 has been previously demonstrated to have a low mutation rate (1 x 10-7 to 1 x 10-8 mutations per p.4 bioRxiv preprint doi: https://doi.org/10.1101/760629; this version posted September 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Kuny et al., manuscript for review August 2019 84 base per infectious cycle), although these studies were performed on a single gene in a unique 85 coding region of the HSV-1 genome [3,4,22]. The HSV-1 genome consists of unique long (UL) 86 and unique short (US) coding regions, which are flanked by large structural repeats (Internal 87 Repeat Long/Short (IRL/S), Terminal Repeat Long/Short (TRL/S). Tandem repeats (TRs) occur 88 frequently in the HSV-1 genome but are especially enriched in the IRL/S and TRL/S regions. 89 Copy number variation or length fluctuations of tandem repeats and homopolymer tracts are a 90 frequent source of genetic variation in strains of HSV-1 [23]. Repetitive regions of the HSV-1 91 genome also have very high G+C content, which favors recombination [21]. Recombination 92 allows for increased genetic diversity in the absence of polymerase error, which may be 93 especially relevant for herpesviruses [21,24,25]. These mechanisms contribute to a high level of 94 variation in the large terminal and internal repeats, which contain genes that are critical to HSV-1 95 replication (ICP0, ICP4, y34.5) [1,26,27]. Crucially, HSV-1 is known to respond quite rapidly in 96 response to strong selective pressures such as antiviral drug treatment, whether through de novo 97 mutation or selection of existing variants in the population [3,28–30].