Host-Dependent Differences in Replication Strategy of The

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bioRxiv preprint doi: https://doi.org/10.1101/2020.03.30.017236; this version posted April 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Host-dependent differences in replication strategy of the Sulfolobus 2 Spindle-shaped Virus strain SSV9 (a.k.a., SSVK1): Lytic replication in hosts 3 of the family Sulfolobaceae 4 5 Ruben Michael Ceballos1,2,3*, Coyne Drummond5, Carson Len Stacy3, 6 Elizabeth Padilla Crespo4, and Kenneth Stedman5 7 8 1The University of Arkansas, Department of Biological Sciences (Fayetteville, AR) 9 2Arkansas Center for Space and Planetary Sciences (Fayetteville, AR) 10 3The University of Arkansas, Cell and Molecular Biology Program 11 4La Universidad Interamericana, Departmento de Ciencias y Tecnología (Aguadilla, PR) 12 5Portland State University, Department of Biology, Center for Life in Extreme 13 Environments (Portland, OR) 14 15 ABSTRACT 16 17 The Sulfolobus Spindle-shaped Virus (SSV) system has become a model for studying 18 thermophilic virus biology, including archaeal host-virus interactions and biogeography. 19 Several factors make the SSV system amenable to studying archaeal genetic mechanisms 20 (e.g., CRISPRs) as well as virus-host interactions in high temperature acidic environments. 21 First, it has been shown that endemic populations of Sulfolobus, the reported SSV host, 22 exhibit biogeographic structure. Second, the acidic (pH<4.5) high temperature (65-88°C) 23 SSV habitats have low biodiversity, thus, diminishing opportunities for host switching. 24 Third, SSVs and their hosts are readily cultured in liquid media and on gellan gum plates. 25 Fourth, given the wide geographic separation between the various SSV-Sulfolobus habitats, 26 the system is amenable for studying allopatric versus sympatric virus-host interactions. 27 Previously, we reported that SSVs exhibit differential infectivity on allopatric and 28 sympatric hosts. We also noticed a wide host range for virus strain SSV9 (a.k.a., SSVK1). 29 For decades, SSVs have been described as “non-lytic” dsDNA viruses that infect species 30 of the genus Sulfolobus and release virions via “blebbing” or “budding” as a preferred 31 strategy over host lysis. Here, we show that SSVs infect more than one genus of the family 32 Sulfolobaceae and, in allopatric hosts, SSV9 does not appear to release virions by blebbing. 33 Instead, SSV9 appears to lyse all susceptible allopatric hosts tested, while exhibiting 34 canonical non-lytic viral release via “blebbing” (historically reported for all other SSVs), 35 on a single sympatric host. Lytic versus non-lytic virion release does not appear to be 36 driven by multiplicity of infection (MOI). Greater relative stability of SSV9 compared to 37 other SSVs (i.e., SSV1) in high temperature, low pH environments may contribute to 38 higher transmission rates. However, neither higher transmission rate nor relative virulence 39 in SSV9 infection drives replication profile (i.e., lytic versus non-lytic) in susceptible hosts. 40 Although it is known that CRISPR-Cas systems offer protection against viral infection in 41 prokaryotes, CRISPRS are not reported to be a determinant virus replication strategy. 42 Thus, the genetic/molecular mechanisms underlying SSV9-induced lysis are unknown. 43 These results suggest that there are unknown genetic elements, resulting from allopatric 44 evolution, that drive virion release strategy in specific host strain-SSV strain pairings. 45 46 * Corresponding Author: Ruben Michael Ceballos, [email protected]; 47

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48 INTRODUCTION 49 50 Reduced model systems have been used extensively to study fundamental properties of 51 virus evolution (Lenski and Levin, 1985; Morgan et al., 2005; Brockhurst et al., 2007). 52 Moreover, it suggested that studying virus-host interactions in reduced model systems may 53 provide opportunities to understand fundamental processes of virus evolution in host 54 systems of agricultural or medical importance (Brockhurst et al., 2007; Dennehy, 2009). 55 Given the recent view that the evolutionary origin(s) of viruses may be linked to the early 56 evolution of Archaea (Forterre et al., 2006; Berliner et al., 2018) and that the emergence of 57 viruses (not bacteriophage) likely pre-dates the divergence of the Archaea and Eukarya 58 (Krupovic et al., 2017; Prangishvili et al., 2017), a robust archaeal virus model system 59 could provide new insights into the evolution of virus lineages and viral replication 60 strategies as well as mechanisms of viral virulence, host resistance, and virus attenuation. 61 62 FIGURE 1. SSV infection of a Sulfolobus host.63 Transmission Electron Micrograph (TEM) of a 30-6064µm epoxy-impregnated section/slice from a SSV-infected65 cell. (60,000X magnification). Vacuole-like anomalies66 in the intracellular space and membrane disruptions are observed in SSV infected host cells. A closer view (inset)67 reveals fusiform or “spinde-shaped” virus-like particles68 on the surface of the cell membrane. 69 70 71 72 73 50nm 200nm 74 75 76 The Sulfolobus Spindle-shaped Virus (SSV) system has become a popular model for 77 studying thermophilic archaeal virus biology and virus-host biogeography. Several factors 78 make this system ideal for studying virus-host infections in crenarchaea (i.e., Sulfolobales). 79 First, endemic populations of SSV hosts from the family Sulfolobaceae exhibit 80 biogeographic structure such that there is a positive correlation between genetic distance 81 among strains (i.e., divergence) and geographic distance between the sites from which 82 strains have been isolated (Grogan et al., 1989; Whitaker et al., 2003; Reno et al., 2009). 83 SSVs also exhibit biogeographic structure on a global-scale (Held and Whitaker, 2009). 84 Second, the highly acidic (pH<4.5) and high temperature (65°C-88°C) SSV-Sulfolobus 85 habitats feature low biodiversity, limiting the potential for host switching, which can 86 confound efforts to elucidate the genetic underpinnings of virus-host infection profiles 87 (Munson-McGee et al., 2018). Third, SSVs and Sulfolobales can be readily cultured both 88 in liquid media (e.g., yeast-sucrose, tryptone) and on gellan gum (e.g., Gel-Rite®) plates 89 (Zillig et al., 1996; Stedman, 2008; Ceballos et al., 2012). Fourth, given the wide 90 geographical separation between the sulfuric geothermal areas, which are habitats for SSVs 91 and their hosts, this system (Fig. 1) is amenable to studying multiple allopatric and 92 sympatric virus-host pairs (Ceballos et al., 2012), an essential for studying virus-host 93 interactions, biogeography, and coevolution (Greischar & Koskella, 2007). 94

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95 For over three decades, SSVs were considered to be non-lytic ‘blebbing’ or budding viruses 96 that exclusively infect Sulfolobus (Martin et al., 1984; Reiter et al., 1987; Palm et al., 1991; 97 Schleper et al., 1992; Zillig et al., 1998; Wiedenheft et al., 2004; Contursi et al., 2006; 98 Prangishvili et al., 2006; Ceballos et al., 2012; Fusco et al., 2015; Quemin et al., 2016). 99 The term non-lytic refers to the fact that SSV infection results in inhibition of cell growth 100 (both in liquid culture and on lawns) rather than the gross lysis of host cells and cell death 101 in liquid culture or clear plaques on host lawns - both of which result from lytic replication. 102 Recently, it was suggested that SSV9 can induce a state of dormancy, empty cells, and 103 eventual host death in a sympatric host; however, the mechanism for this proposed 104 dormancy is unclear and there does not seem to be any cell lysis (Bautista et al., 2015). 105 106 Plaque-like “halo” assays using both sympatric and allopatric hosts have repeatedly 107 demonstrated that different SSVs (e.g., SSV1, SSV2, SSV3, SSV8, SSV10) form turbid 108 indentations or halos on host lawns (Fig. 2A, B), which often feature diffuse boundaries 109 (Martin et al., 1984; Schleper et al., 1992; Wiedenheft et al., 2004; Ceballos et al., 2012; 110 Iverson and Stedman, 2012). Yet, halo assays using SSV9 (formerly known as SSVK1), 111 isolated from the Valley of Geysers (Russian: Долина гейзеров) Kamchatka, Russia 112 (Wiedenheft et al., 2004), form large clear plaques on host lawns (see Fig. 2C) in contrast 113 to the turbid diffusely-bound halos characteristic of all other SSVs (Ceballos et al., 2012), 114 indicating that SSV9 may replicate differently than the other SSVs (Bautista et al., 2015). 115 116 FIGURE 2. SSV Spot-On-Lawn Halo Assays. A 1-2µL drop of virus suspension 117 was spotted on lawns of DSM 1617 (formerly, Sulfolobus solfataricus strain P2) to determine whether a viral infection can be established and the nature of the virus-host 118 interaction in terms of halo “phenotype”. (A) SSV1 forms broad diffuse, turbid halo. 119 (B) SSV8 (a.k.a., SSVRH) makes a broad diffuse, turbid halo with a sharper boundary 120 between the direct application point and halo leading edge; (C) SSV9 (a.k.a., SSVK1) 121 yields a large and complete clearing of host lawn. 122 123 124 125 126 To test the hypothesis: SSV9 lyses susceptible hosts of the family 127 Sulfolobaceae - host growth in single-virus/single-host interactions 128 using three distinct SSV strains (i.e., SSV1, SSV8, and SSV9) was 129 evaluated in both liquid culture and with spot-on-lawn “halo” assays. 130 Both allopatric and sympatric hosts of the family Sulfolobaceae 131 (Order: Sulfolobales) were used to determine relative susceptibility. 132 Several different liquid culture conditions were used, specifically: a 133 variety of distinct virus strain-host strain pairings were examined; 134 different multiplicities of infection (MOI) were tested; and, end-point 135 assays measuring virus and host dynamics in parallel were conducted. 136 Spot-on-lawn halo assays and observations from small-scale liquid culture infections were 137 conducted to examine plaque-like halo formation and the relative amount of cell debris 138 emerging from different virus-host strain pairings, respectively. The relative stability of 139 different SSV strains under conditions that simulate the natural environment of these 140 viruses and their hosts were also conducted to assess whether virion stability plays a role 141 in successful transmission (and thus between strain differences in virion production).

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142 RESULTS 143 144 Virus Yields and Confirmation of SSV Infection 145 146 Titers of virus stocks were determined by one (or more) of four distinct methods: 147 electrospray ionization/mass spectrometry (ESI/MS) in units of virus particles per milliliter 148 (VP/mL); serial dilution plaque-like assays in units of halo-forming units per milliliter 149 (hfu/mL); virus-like particle (VLP) counts using transmission electron micrographs 150 (VLPs/mL); and/or, quantitative polymerase chain reaction (qPCR) in terms of the total

151 number of viral genomes per milliliter (vg/mL). For infection assays, end-point titers were 152 also determined by one or more of these methods with ESI/MS and plate assays preferred. 153 154 Growing 600mL liquid cultures of permissive host, each infected with a single SSV strain 8 155 (i.e., SSV1, SSV8, or SSV9), to an optical density (OD600nm) of 0.8 (~5.4x10 cells/mL) 156 results in comparable virion yields as shown by electrospray ionization/mass spectrometry 157 (ESI/MS) spectra (Fig. S1). Upon: harvest, centrifugation, removal of supernatant, 158 supernatant filtration (0.45µm) and concentration by ultrafiltration (~300-fold) - SSV8 and 159 SSV9 suspensions result in ~2x108 – 4x108 VP/mL. SSV1 yields are typically 50-80% 160 lower. Thus, it takes 1.2-2.0L of SSV1-infected culture to ensure stocks are of the same 161 order of magnitude and comparable to SSV8 and SSV9. SSV suspensions were then 162 diluted to produce working stocks with equivalent titers (e.g., ~1x108 VP/ml). These 163 working stocks were used for comparative infection assays with the same multiplicity of 164 infection (MOI). MOIs of 0.01 and 0.001 were typically used but other MOIs (e.g., 165 MOI=3) were also tested. 166 167 It has previously been reported that not all species within the family Sulfolobaceae are 168 susceptible to SSV infection (Ceballos et al., 2012). Thus, to ensure that there was active 169 infection in the virus-host pairings selected for this study, three steps were taken. First, 170 virus suspensions were examined under transmission electron microscopy (TEM) to ensure 171 that spindle-shaped virus particles (i.e., virions) were present in the harvested virus stocks. 172 Second, spot-on-lawn plate assays were used as a qualitative (i.e., yes or no) verification 173 that a given SSV strain is able to infect a specific strain within the family Sulfolobaceae. 174 Third, post-infection titers and TEM were performed to verify active infection (Fig. 3). 175 176

177 178 FIGURE 3 – Transmission Electron Micrographs of SSV Particles. Transmission Electron Microscopy 179 (TEM) demonstrates the presence of spindle-shaped virus particles after harvest and concentration steps at end- 180 points of infection assays as well as during the preparation of virus stocks for liquid culture infection assays and 181 halo assays. TEM images of SSV1 [left], SSV8 [middle], and SSV9 [right].

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182 Host-Growth after Infection by Various SSVs 183 184 Liquid culture assays using several SSVs infecting the universally susceptible host strain 185 Sulfolobus strain Gq (Cannio et al., 1998; Ceballos et al., 2012), reveal that host growth

186 (µmax) is slowed and the “carrying capacity” upon entering stationary phase (i.e., Nasymptote) 187 is reduced compared to uninfected control (Fig. 4). Individual (i.e., single virus-single host) 188 infections with SSV1, SSV8, or SSV10 on strain Gq show archetypal host growth with 189 these reportedly non-lytic SSVs. Infection with either of two SSVs isolated from two 190 geographically-distinct Icelandic geothermal regions - SSV2 (from Reykjanes) and SSV3

191 (from Krisovic) - also show reductions in µmax and area-under-the curve (AUC) when 192 compared to controls. Despite SSV2 and SSV3 infections exhibiting slightly different host 193 growth profiles (Fig. 4A, cyan and mustard lines) due to slower growth to stationary phase, 194 data still fit to a Gompertz model with high coefficient of determination values (Table S1). 195 Measures of percent inhibition can be derived by area-under-the-curve (AUC) calculations 196 for the infected host growth and dividing by the AUC for the uninfected control (Fig. 4B). 197 Using this quotient, an index of relative virulence (VR) between strains on a given host can 198 be generated (Stacy and Ceballos, 2020). 199

200 201 FIGURE 4 – Growth of host strain Sulfolobus Gq during SSV infection. (A) Sulfolobus strain Gq 202 challenged with SSV1 (Palm et al., 1991), SSV2 (Stedman et al., 2003), SSV3 (Stedman et al., 2006), 203 SSV8 (a.k.a., SSVRH; Wiedenheft et al., 2004) and SSV10 (a.k.a., SSVL1; Goodman & Stedman, 2018). 204 (B) Graphical representation of percent inhibition of SSV1 and SSV8 using area-under-the-curve (AUC). 2 205 All growth curves fit with high coefficients of determination (R values) to Gompertz Models or Logistic Growth Models (see Table S1). The asymptotes (dashed) indicate point of inoculation until end of the 206 exponential growth phase/onset of stationary phase. Infections were conducted at an MOI = 0.01. 207 208 209 Using a Gompertz model and AUC as a measure of percent inhibition (PI), SSV1 exhibits 210 the lowest PI (8.57%) while SSV8 has a significantly higher PI (28.44%) with R2 values 211 for the Gompertz of 0.9875 and 0.9775, respectively. Employing Riemann Sums or

212 alternative models (i.e., Logistic Growth) does not significantly alter relative virulence (VR) 213 for SSV1 and SSV8 (see Table S1). Regardless of analytical approach, the general trend in 214 virulence of SSVs on Gq is: SSV1

218 219 Host-Growth after Infection with SSV9 220 221 Unlike infection with SSV1, SSV2, SSV3, SSV8 or SSV10, infecting Sulfolobus strain Gq 222 with SSV9 did not grow with Gompertzian-like (Gompertz, 1825; Laird, 1965) dynamics. 223 In contrast, rapid and significant inhibition of culture growth was observed in cultures 224 infected with SSV9. This pattern was observed for several strains of Sulfolobales over 225 multiple trials with each strain (Fig. 5). Host strains tested were isolated from 226 geographically-distinct geothermal regions from around the world and serve as allopatric 227 hosts to SSV9. 228

229 230 231 FIGURE 5 – Growth Profiles for Susceptible Allopatric Host Strains Infected with SSVs. Under equivalent Multiplicity of Infection (MOI=0.1), allopatric hosts show classic Gompertzian growth when 232 infected with either SSV1 or SSV8. However, cultures of the same hosts infected with SSV9 exhibit 233 drastic growth inhibition. Growth in SSV9-infected cultures are “saw-toothed” with damped oscillations.

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234 Liquid culture assays using SSV1, SSV8, and SSV9 on susceptible allopatric host strains: 235 S437 (DSM1617 from the Deutsche Sammlung von Mikroorganismen und Zellculturen), 236 a type strain (a.k.a., S. solfataricus P2) isolated from a hot spring near Pisciarelli, Italy; 237 S200 (a.k.a., S. icelandicus HVE 10/4 from the Hveragerdi thermal region of Iceland); and, 238 S444 (an isolate derived from Lassen Volcanic National Park, California, USA) – show

239 reduced Nasymp, AUC, and/or µmax for SSV1 and SSV8 (versus uninfected controls). 240 However, when each host was infected with SSV9, a distinct host growth profile emerged. 241 Specifically, Gompertzian Models failed to adequately represent the SSV9 infection data. 242 Instead, non-Gompertzian cyclical spike patterns are consistently observed in host growth. 243 Low R2 values (<0.5) emerged for both individual traces and average curves for SSV9, 244 whereas, high R2 values (> 0.9) were typical for all other SSVs infecting these same hosts 245 (Table S2). 246 247 Host-Growth Profile of SSV9 is not MOI-Dependent 248 249 To determine whether the unique growth profile of SSV9-infected hosts was dependent on 250 MOI, dilutions of SSV8 and SSV9 stocks were prepared and liquid culture infection assays 251 were performed on Sulfolobus strain Gq. The typical host growth pattern shown for SSV 252 infection (i.e., Gompertzian-like growth) is observed for all trials of SSV8 infection on 253 strain Gq regardless of MOI used (Fig. 6A). A positive correlation between SSV8 MOI 254 and percent inhibition of host growth was generally observed. However, for the lowest 255 MOI (1:64 and 1:256 dilutions), the PI was not significantly different from uninfected 256 control despite productive infection. The atypical host growth profile for host infected with 257 SSV9 also persisted (Fig. 6B). Amplitudes of host growth spikes change with an inverse 258 relationship in response to MOI; however, there was no switch to the Gompertzian-like 259 effect even at the lowest SSV9 MOI.

260 261 FIGURE 6 – Growth of Sulfolobus strain Gq Infected at Different MOIs: SSV8 versus SSV9. 262 Under different Multiplicity of Infections, host strain Gq (a.k.a., S13) infected with SSV9 retains the 263 cyclical “saw-toothed” profile characteristic of SSV9 infection (and typical of lytic virus systems). Under 264 different MOIs (1:1 represents a MOI = 0.1), strain Gq infected with SSV8 retains Gompertzian growth. 265 266

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267 268 End-Point Infection Assays Indicate SSV9 Lytic Replication 269 270 To examine both host and virus population dynamics in a liquid culture infection assay, 271 two large-scale trials (i.e., 14+ replicates) at higher observation frequency (every 4 hrs) 272 were conducted with Sulfolobus strain Gq infected with SSV8 and SSV9 at an MOI = 1 273 (Fig. 7). Two flasks were harvested every 12 hrs from the suite of replicates to determine 274 virus particle counts via ESI/MS. Sulfolobus strain Gq infected with SSV8 showed typical 275 Gompertzian-like host growth with a concomitant increase in virus particle count (Fig. 7A) 276 was observed as the SSV8 infection continued through 78 hours post-infection (HPI). 277 SSV9-infected Sulfolobus strain Gq showed a distinct pattern from the Gq-SSV8 infection. 278 SSV9-infected growth peaked (18 HPI) followed by a rapid drop in cell density (Fig. 7B). 279 The decrease in host density was followed by a spike in SSV9 particle count (32 HPI). 280 A B Gq vs. SSV8 Gq vs. SSV9

281 282 FIGURE 7 – Virus-Host Dynamics: SSV8 versus SSV9 infections in Sulfolobus strain Gq. Using a 283 high multiplicity of infection (MOI=1) to ensure virus particle detection via ESI/MS, strain Gq was 284 infected with SSV. (A) Sulfolobus strain Gq at an OD600 = 0.15-0.20 is inoculated with SSV8 at MOI=1 285 resulting in standard Gomperz-like culture growth with a concomitant increase in virion count over time.

286 (B) Sulfolobus strain Gq at OD600≈0.15-0.20 is inoculated with SSV9 at MOI=1 resulting in an average 287 peak density of OD600≈0.45 followed by a decrease in cell density and a sudden spike in SSV9 particle 288 count, peaking at 32 HPI. Cell density stabilizes at OD600≈0.30-0.35 from 60-90HPI. SSV9 particle 6 6 289 count drops to the detectable limits of the ESI/MS (approximately 1x10 -2x10 virus particles/mL). 290 291 Interestingly, other than the initial peak in host cell density, no noticeable subsequent peaks 292 (dampened or accentuated) were observed in this trial with the higher MOI (see Fig. 7B). 293 It was also noted that the growth inhibition did not result in an optical density value of zero. 294 Instead, cell density stabilized at approximately one-third the average peak value. 295 Furthermore, SSV9 titer (via ESI/MS) decreased substantially (see Fig. 7B, 60-100HPI). 296 Although the general growth profiles were consistent with prior trials for non-lytic versus 297 lytic release, these latter two features were not expected (see Discussion). Together, these 298 data indicate that SSV8 pursues canonical non-lytic virion release expected of SSVs, while 299 SSV9 lyses host strain Gq. This is also supported qualitatively by the presence of 300 significantly more cell debris in SSV9-infected small-scale infection assays (Fig. S2). 301

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302 SSV9 Lytic Replication may be Limited to Allopatric Hosts 303 304 To determine if SSV9 lytic replication was dependent upon host strain allopatry, two 305 isolates of Sulfolobales derived from volcanic hot springs in Kamchatka, Russia were also 306 tested as plausible hosts for SSV9. Isolate S147 was derived from the same geothermal 307 area in Geyser Valley as the original host from which SSV9 was isolated. Isolate S150 was 308 derived from the Mutnovsky volcanic region near the south tip of the Kamchatka peninsula. 309 To determine how SSV9 infection would impact growth on a sympatric host (S147) and a 310 quasi-sympatric host (S150), spot-on-lawn and liquid culture infection assays were 311 conducted with these two isolates. Spot-on-lawn assays indicate that SSV9 does not form 312 halos on S150 lawns; however, halos readily form on lawns of S147 (Ceballos et al., 2012). 313 Liquid culture-based infection assays substantiate S150 resistance to infection (Fig. 8A) 314 and confirm that S147 is susceptible to SSV9. Post-infection titers for the S150 infection 315 trials did not show any detectable virus; while those for S147 showed virus particle counts 316 on the order of 109 VP/mL indicating productive infection (Fig. S4). The most striking 317 result was that S147 growth during SSV9 challenge resembles the non-lytic archetype 318 infection dynamics observed with other SSVs (Fig. 8B). 319 320 AUC analysis for S150-SSV9 trials results in no significant growth inhibition (PI < 0.05), 321 while the S147-SSV9 trial shows a significant PI (>0.20) with a convincing Gompertz fit 322 (R2 = 0.934), indicating that SSV9 exhibits a non-lytic replication profile on a sympatric 323 host, S147 (see Table S3). 324

325 326 FIGURE 8 – Impacts of SSV9 challenge on Isolates from Geothermal Regions in Kamchatka. 327 Isolates of Sulfolobales from geothermal regions of the Kamchatka peninsula were infected with SSV9. 328 Isolate S147 was derived from the same hot spring region as the host from which SSV9 was derived. S147 is considered a sympatric host. Isolate S150 was also derived from the Kamchatka peninsula but 329 from a geothermal region approximately 256 km to the southwest of Geyser Valley. S150 is considered 330 quasi-sympatric. (A) Strain S150 growth curves for SSV9-infected culture exhibit no significant changes

331 in µmax compared to the uninfected control curves. (B) Strain S147 growth curves for SSV9-infected 332 cultures resemble canonical non-lytic replication. 333 334

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335 DISCUSSION 336 337 Previously published work demonstrated that some Sulfolobales are completely resistant 338 to infection by well-characterized SSVs (i.e., SSV1, SSV2, SSV3, SSV8, SSV9, SSV10) 339 while others are susceptible to subset or all of these SSVs in spot-on-lawn “halo” assays 340 (Ceballos et al., 2012). Large plaque-like halos were observed on host lawns challenged 341 with SSV9 (at the same MOI as other SSVs) suggesting that SSV9 is more virulent. 342 However, the large clearings on host lawns generated by SSV9 appeared to be true plaques 343 rather than the turbid halos characteristic of SSV infection. This raised the possibility that 344 SSV9 is lysing host rather than using non-lytic “blebbing” as a means of virion release. 345 Although the halo assay is sufficient for determining whether a given host is susceptible 346 (or not) to a specific SSV, it offers limited information regarding the dynamics of infection. 347 In the present study, liquid culture assays and host growth curve analyses were used to test 348 the hypothesis that: SSV9 employs a lytic replication strategy rather than the canonical 349 non-lytic replication (i.e., virus blebbing) characteristic of other SSVs. 350 351 SSV infection in liquid culture. Growth curve analysis provides insights into the nature of 352 virus-host infection dynamics that are not resolvable through plate-based halo assays. 353 Using host growth curve analyses, it is not only possible to determine percent inhibition 354 and relative virulence of a different SSVs on a host (see Fig. 4A) but it is also possible to 355 gain insights into rates of virus replication/transmission as well as virion release strategy. 356 By measuring titers of inocula at the beginning of an infection trial, establishing equivalent 357 MOI for different virus-host pairings, and taking end-point titers, a quantitative assessment 358 of how many virus particles are present in the culture at the time of inoculation versus how 359 many progeny virions are present at the end-point is possible. However, this method of 360 comparing pre-infection titer and post-infection titers may be confounded by differential 361 stability of distinct SSV strains in the high temperature (76-80°C) and low pH (3.0-3.4) 362 culture environment. Although SSV8 and SSV9 appear to be more resilient in solution, 363 SSV1 does not seem to be as stable (Fig. S3). Thus, viral fecundity may be underestimated 364 by virus count since some virus particles (e.g., SSV1 virions) breakdown during the course 365 of the infection assay. Still, end-point virus count is needed to confirm productive infection. 366 Specifically, if the number of virus particles at end-point exceeds the number of virus 367 particles used for inoculation, then this indicates that the infection was productive. 368 However, if virus count at end-point is equal to or less than virus particle count at 369 inoculation, then it can be argued that no infection occurred. Although it can be argued 370 that a highly unstable virus strain could rapidly breakdown yielding a false negative for 371 infection, TEM (see Figs. 1 and 3) and spot-on-lawn halo assays (see Fig. 2) are used as 372 secondary methods to validate host susceptibility/SSV strain infectivity. 373 374 Archetypal non-lytic SSV replication and host growth profiles. Despite limitations in 375 determining end-point virus count due to differences in virion stability between the 376 different SSVs, liquid culture assays permit a quantitative assessment of relative virulence

377 (VR) by calculating percent inhibition (PI) of each SSV on a given host strain (see Fig. 4B). 378 The archetypal non-lytic replication strategy of SSVs allows quantitative assessment of VR

379 based on comparisons between Nasymptote, µmax, and/or AUC in the SSV-infected versus the 380 uninfected control states.

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381 Atypical infection dynamics of SSV9 on allopatric host growth. Archetypal SSV infection 382 (see Figs. 4) induces Gompertz-like host growth profiles (Stacy and Ceballos, 2020), 383 indicative of non-lytic infection, which in turn is consistent with halo, instead of plaque, 384 formation on spot-on-lawn plate assays. However, when SSV9 is used to infect a series of 385 susceptible allopatric hosts, atypical infection dynamics emerge in the host growth curve. 386 Specifically, a deep inhibition, which manifests as “saw-tooth” or “serial spike” profile in 387 the host growth curve, occurs suggesting a different replication process (Fig. 5, red traces). 388 Although it may be suggested that SSV9 is simply more virulent on the susceptible host, 389 this argument is refuted by the fact that neither a Gompertz or Logistics growth models fit 390 reasonably to the SSV9-infection host growth data (as indicated by very low R2 values). 391 392 Atypical infection dynamics of SSV9 are not MOI dependent. Further support for unique 393 SSV9 infection dynamics rests in the inability to elicit the archetypal non-lytic growth 394 profile by manipulating MOI (see Fig. 6B). Even under low MOI, SSV9 continues to 395 induce a cyclic spike profile in the host growth curve. Likewise, varying MOI for SSV8 396 does not change the archetypal non-lytic growth profile to one resembling SSV9 infection. 397 To determine if a change in the SSV8-infect host growth profile could be forced by a 398 concentrated inoculum, a 20:1 virus concentrate was prepared. Although the culture 399 exhibited a strong depression (data not shown), the curve still fit a Gompertz Model with 400 a high coefficient of determination (R2 = 0.87) and did not exhibit a cyclic spiking profile. 401 Furthermore, virus particle count at the end of the 20:1 viral concentrate trial was 2-fold 402 greater than what was after the initial inoculum, indicating that the depression in the growth 403 curve was not due to lysis-from-without (Delbrück, 1940) but due to productive infection. 404 405 SSV population dynamics support lytic replication. Larger-scale liquid culture assay trials 406 (i.e., greater number of replicate flasks) allow replicates to be harvested periodically during 407 the course of infection to monitor changes in virus titer in parallel to host growth (Fig. 7). 408 Sampling at shorter intervals (i.e., every 4 hrs), infection with SSV8 (a non-lytic replicator) 409 shows an increase in virus particle count concomitant with host growth. However, SSV9 410 infection of the same host strain (i.e., Gq) shows sharp peak in virus particle count ~12 hrs 411 after a peak and a notable rapid drop in host cell density. This is consistent with lytic virus 412 release or a “burst” event (Fig. 7B). Interestingly, even though the infection assay was 413 carried out to ~98 HPI, no subsequent “peak-and-crash” of either host (or virus) occurred 414 after the first cycle at 20-32 HPI. Moreover, optical density (a proxy for host cell density)

415 remained steady at OD600nm≈ 0.3 from 60-98 HPI. The reason underlying the stability of 416 host cells with no additional SSV9-induced lysing or SSV9 virion production is not clear. 417 There is one report suggesting that SSV9 challenge may induce dormancy in viable hosts

418 (Bautista et al., 2015), which may be the reason for sustained absorbance at OD600nm. 419 Indeed, dormant cells would not likely support virus production and may render cells 420 resistant to infection. Another report suggests that group II chaperonin complexes may 421 form highly stable networks of chaperonin complex filaments at the intracellular surface 422 of archaeal cells (Trent et al., 2003). Cell membrane-associated HSP complexes as either 423 filaments or two-dimensional arrays can maintain cell shape even if cells are not viable. 424 Under scanning electron microscopy (SEM), cells from infected culture, which were likely 425 non-viable “ghost cells” due to presence of large holes in the cell membrane, maintained a 426 round, lobed three-dimensional structure that could absorb 600nm light (data not shown).

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427 Whether the absence of subsequent cycles of host recovery, SSV9 infection, and lytic 428 bursting beyond the initial cycle are due to cells becoming dormant or whether these are 429 “ghost” cells requires further study. Nonetheless, host growth curves for SSV9-infected 430 strains display atypical profiles that do not fit to Gompertzian (or Logistic Growth Models) 431 as is expected for non-lytic virion release but rather represent lytic bursts of virion release. 432 Thus, it is clear from both spot-on-lawn and liquid culture assays that SSV9 is lysing host. 433 Small-scale (102 µl tube) infection assays further support this conclusion. Specifically, 434 infection with SSV9 generates significantly more visible cell debris at the same time point 435 of infection compared to infection with other SSVs (see Fig. S2), indicating cell lysis. 436 437 SSV9 lytic replication may depend on host allopatry. Although SSV9 lytic behavior does 438 not appear to be dependent upon MOI and seems to be an inherent property of the SSV9 439 replication strategy, all susceptible host initially tested were allopatric hosts isolated from 440 geothermal hot springs thousands of kilometers away from where SSV9 was isolated. Yet, 441 when two Sulfolobales – one from the same geothermal region from which the original 442 SSV9 was isolated and another from a distant spring (ca. 250 km) within the same area 443 (i.e., Kamchatka, Russia) – were challenged with SSV9, unanticipated dynamics emerged. 444 The quasi-sympatric S150 strain showed no susceptibility to SSV9 in spot-on-lawn assays. 445 Moreover, in liquid culture assays with S150, there were no significant differences in

446 Nasymptote, µmax, or AUC between the SSV9-infected and S150 uninfected controls (Fig. 8A). 447 This is not surprising since other Sulfolobales are reported to be resistant to SSV infection. 448 However, when SSV9 was used to challenge the sympatric isolate S147, there were 449 unexpected results. Specifically, it appears that SSV9 does not only infect S147 but that 450 the infection follows canonical non-lytic replication characteristic of other SSVs (Fig. 8B). 451 452 Recently, a series of reports have proposed a re-organization in the taxonomy of the family 453 Sulfolobaceae to include four distinct genera: Sulfolobus (Brock et al., 1972), Stygiolobus 454 (Segerer et al., 1991), Sulphurisphaera (Kurosawa et al., 1998), and the most recently 455 proposed genus Saccharolobus (Sakai and Kurosawa, 2018; Tsubio et al., 2018). Whether 456 SSV infectivity and relative virulence is correlated with these newly defined genera 457 remains to be determined. Our lab has demonstrated that SSVs infect select strains 458 belonging in at least two of these genera. Although this re-organization of the phylogenetic 459 tree for the family Sulfolobaceae requires the community to revisit previously published 460 work, the non-lytic growth profile observed in the S147-SSV9 infections raises a question 461 of whether sympatric versus allopatric virus-host evolution determines replication strategy. 462 Since lytic replication typically results in greater virulence (than non-lytic replication), 463 there is also the question of whether a switch in replication strategy over evolutionary 464 timescales (i.e., lytic to non-lytic) is part of a more generalizable pattern of attenuation in 465 the virus-host coevolutionary “arms race” (Van Valen, 1973; Dawkins and Krebs, 1979). 466 Furthermore, the genetic substrates underlying the emergence of distinct replication 467 strategies in SSV systems remain unknown. Employing high throughput sequencing 468 methods (e.g., nanopore sequencing) and advanced analytical techniques may resolve the 469 impacts of specific genetic factors on virus-host dynamics and quantify relative virulence 470 between non-lytic versus lytic phenotypes, respectively (Stacy and Ceballos, 2020), may 471 help to address the aforementioned questions and identify genetic elements that predispose 472 viruses to lytic versus non-lytic replication strategy.

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473 MATERIALS AND METHODS 474 475 Virus preparation. Glycerol stocks of SSV-infected Sulfolobus strains stored at -80°C 476 were partially thawed on ice. For most trials, 100μL of infected cell suspension was added

477 to 30mL of YS media (3.0g/l (NH4)2SO4, 0.7g/l glycine, 0.5g/l K2HPO4, 2.0g/L sucrose, 478 1.0g/l Yeast Extract, 200.0μl/l 1% FeSO4•7H2O, 244.0μl/l 1% Na2B4O7•10H2O, 90.0μl/l 479 1% MnCl2•4H2O, 11.0μl/l 1% ZnSO4•7H2O, 2.5μl/l 1% CuSO4•5H2O, 1.5μl/l 1% 480 Na2MoO4•2H2O, 1.5μl/l 1% VOSO4•5H2O, 0.5μl/l 1% CoCl2•6H2O, 0.5μl/l 1% 481 NiSO4•6H2O, 1.0μl/l 1.0M MgCl2/0.3M Ca(NO3)2 solution; pH adjusted to 3.2 with H2SO4) 482 in a 125mL Erlenmeyer flask. Alternatively, T media was used in select experiments using 483 the same solution but with 3.0g/l tryptone substituted for the yeast and sucrose components. 484 Flasks were partially sealed to avoid evaporation and incubated at 76-78°C/70-90 RPM.

485 Once liquid culture reached an optical density (OD600) between 0.4 and 0.6, 3.0mL of cell 486 suspension was used to inoculate 600mL of pre-heated fresh media in a 1.0L baffled flask.

487 These cultures were incubated at 78°C/70 RPM until reaching an OD600 = 0.6-0.8 to 488 maximize virus yields. All 600mL of the culture was centrifuged for 20 min at 6,000 RPM 489 (Sorvall RT Legend Centrifuge, Fiberlite 4x8000ml fixed-angle rotor with 250ml inserts; 490 ThermoFisher, Pittsburgh, PA) to pellet the cell mass while leaving virus in suspension. 491 The upernatant was decanted and filtered through a 0.45μm vacuum PES filter system. 492 The filtrate was concentrated using 10kDa Centricon Plus-70 spin concentration tubes 493 (Millipore Corp., Billerica, MA, USA) to produce ~3.0mL concentrated SSV suspension. 494 TEM was used to confirm the presence of virions. Virus particle count was measured using 495 electrospray ionization/mass spectrometry or serial dilution plaque-like assays (see below). 496 Virus was stored at 4°C and used in spot-on-lawn “halo” assays and liquid culture infection 497 assays within 2-3 weeks of harvesting. Dilutions of viral stocks were used as inocula. 498 499 Host cell preparation. Glycerol stocks of uninfected Sulfolobus strains stored at -80°C 500 were used to establish 30mL cultures in YS (or T or TS) media, as described above. 501 Uninfected Sulfolobus cultures were either used to prepare host lawns for halo assays 502 (Ceballos et al., 2012) or used in liquid culture infection assays (as described below). 503 504 Transmission electron microscopy (TEM) of viruses and cells. To verify the presence of 505 SSVs in samples, ~5μL of viral suspension was spotted onto a formvar-coated copper grid 506 and incubated for 10 min in a humidity chamber. The sample was rinsed off with distilled 507 water and negatively stained with a 1% solution of uranyl acetate for 30 seconds. The stain 508 was wicked and the sample was air dried. Grids were imaged with a Hitachi H-7100 TEM 509 at 75kV. Images were captured at 60,000-200,000X magnification. TEM images for cells 510 were acquired as described in Brumfield et al. (2009). Cells were fixed in glutaraldehyde 511 (3% v/v), centrifuged, and re-suspended in a small volume of agar (2% v/v). After 512 solidification, the resulting agar was cut into small pieces and fixed overnight with 513 glutaraldehyde (3% v/v) in 0.05M potassium sodium phosphate buffer (PSPB) and pH=7.2. 514 Agar pieces were rinsed twice for 10 min each with PSPB. Agar pieces were post-fixed 515 with osmium tetroxide (2% v/v) at RT for 4h. Samples were dehydrated via an ethanol 516 rinse series (50%-100% v/v) and then washed with a transitional solvent, propylene oxide. 517 Spurr’s resin (Spurr, 1969) was used to infiltrate dehydrated cell mass and pieces were 518 baked overnight at 70ºC. Thin sections (60-90 nm) were cut and stained with uranyl acetate 519 and lead citrate. Imaging was done with a LEO 912AB TEM.

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520 Electrospray ionization/mass spectrometry (ESI/MS). Virus suspension preparations were 521 processed in an Integrated Viral Detection System (BVS, Inc.; Missoula, MT) per protocols 522 in Wick et al. (1999). In brief, a mixture consisting of 100μL viral suspension and 900μL 523 ammonium acetate buffer solution is aerosolized in a Electrospray Aerosol Generator 524 (Model 3480, TSI, Inc., Minnesota). A Differential Mobility Analyzer (Model 3081, TSI, 525 Inc., Minnesota) separates particles by their electrical mobility, which is influenced by 526 particulate mass-to-charge ratio or “size”. These particles flow in tandem with a saturated 527 butanol fluid. The particles initiate butanol condensation and the stream is cooled enabling 528 butanol-condensed particles to be optically counted in a Condensate Particle Counter 529 (Model 3772, TSI, Inc., Minnesota). System software displays results in terms of particle 530 count per size category with a standard range of 2-280nm. This specialized ESI/MS is 531 designed to detect intact virus particles (Wick et al., 2006) and can measure relative virus 532 particle count between two or more samples. In these infection assays, ESI/MS spectra 533 were evaluated by using either peak values or the area under the curve to compare SSV 534 particle production between two cultures. 535 536 Note: Time-of-flight estimation based on mass:charge is used to calculate ESI/MS “size”. 537 ESI/MS “size” will differ from physical size as measured by other methods such as 538 transmission electron microscopy. In addition, mass:charge size versus physical size will 539 differ due to the assumption of virus particle sphericality in time-of-flight derivations. 540 SSVs have been shown under electron microscopy to be fusiform or “spindle-shaped” 541 particles of approximately 60 x 90 nm (and not spherical), thus, a discrepancy is expected. 542 In some cases, SSVs exhibit dual peaks or shouldered peaks under ESI/MS. Such spectral 543 features may be due to different conformations of the same SSV or, potentially, due to the 544 presence of pleomorphic SSVs. Under ESI/MS, SSVs exhibit characteristic peaks between 545 46.1 and 61.5 nm for purified virus suspensions with major peaks typically appearing at 546 46.1, 47.8, and 49.6 within the ±4nm system tolerance. 547 548 Spot-on-lawn halo assays. Halo assays were performed as in Stedman et al. (2003).

549 Cultures were grown as described above. At an OD600= 0.4-0.6, 500μL of cell suspension 550 was added to 4.5mL of a 78°C mixture of equal parts 1.0% w/v Gelrite® (Sigma-Aldrich, 551 St. Louis, MO, USA) and two-fold concentrated YS medium. The 5mL mixture was 552 spread on pre-warmed Gelrite® plates (1% w/v in medium) and allowed to solidify for 15 553 minutes at room temperature followed by a 20 min incubation at 78°C. 1.0μL of each viral 554 suspension was spotted onto the prepared plate in labeled areas. Depending upon host 555 strain, plates were incubated for 3-9 days at 78°C. Successful infection was scored by the 556 formation of a visible halo of growth inhibition on the host lawn. Triton X-100 (0.05% v/v) 557 was used as a positive control and sterile water was used as a negative control. 558 559 Liquid culture infection assays. As described above, 3.0mL of uninfected Sulfolobus 560 culture was diluted 1:100 in pre-heated media in a 1.0L baffled flask. Cultures were grown

561 to an OD600≈ 0.15. Then, 100μL of standardized viral suspension was added to the flask. 562 Sulfolobus cultures were incubated with shaking (78°C/70RPM) for various time intervals. 563 Virus was then harvested as detailed above and ESI was used for virus particle counts. 564 Growth curves from SSV-Sulfolobus cultures were fit to: Logistic and modified Logistic 565 (eqs. 1,2); Gompertz and modified Gompertz (eqs. 3, 4); and, other growth models 566 (Gompertz, 1825; Laird, 1964; Lopez et al., 2004, Zwietering et al., 1990; Sprouffske & 567 Wagner, 2016): 14 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.30.017236; this version posted April 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

568 569 �(�) = (equation 1) ( ) 570 571 �(�) = (equation 2) ( () 572 573 �(�) = A⋅ exp [−ln( ) ⋅ exp(−� ⋅ �)] (equation 3) 574 ⋅() 575 �(�) = A⋅ exp [−exp( (λ−t)+1)] (equation 4) 576 577 The exp represents an exponential function such that exp(x)=ex, where e is Euler’s number. 578 A represents the amplitude or peak growth value in the given environment, which 579 corresponds to stationary phase and the maximum carrying capacity; � is the intrinsic

580 growth rate coefficient; �0 is the initial population size; μ is the maximum slope of the 581 growth curve, and t is time. For equations in which the maximum specific growth rate, μ

582 or μmax, was not a fit parameter; it was calculated by taking the derivative of y(t) evaluated

583 at a software-derived point of max growth using parameter estimates for A, �, and �0. 584 585 Parameter estimates were derived in R 3.6.1 (R Core Team; R Foundation for Statistical 586 Computing, Vienna, Austria) using curve fitting functions of easynls, growthcurver, and 587 grofit packages (Arnhold, 2017; Sprouffske & Wagner, 2016; Kahm et al., 2010). The best 588 parameter values for each function were found via Levenberg-Marquardt non-linear least- 589 squares algorithm (Moré, 1978). The levels of fit between software packages varied and 590 provided varied slightly different parameter values for each dataset. Relative area under 591 the curve values; however, remained consistent across assessments within the same fitted 592 models (i.e. Gompertz and Gompertz with software “A”) and with direct calculation of 593 Riemann sums of integral of curves. The area under the optical density-based growth 594 curves was calculated via two methods for each data set. The fitted models were integrated 595 with from time t = 0 to the time-point corresponding to the end of the exponential growth 596 phase of the uninfected host-control’s growth curve. This process was utilized for each data 597 set was able to be successfully fit to equations 1-4 above, as well as other growth models 598 not shown here. The second method for calculating AUC values was through Riemann 599 sums, also known as the trapezoidal approximation method (Hasenbrink et al., 2005) 600 Equation 5 shows the formula utilized for calculating the AUC with this method. 601 602 �� = × (� − �) (equation 5) 603 604 Area under the curve values for virally challenged hosts were then standardized to the AUC 605 value for the averaged uninfected host control growth curve in order to calculate the percent 606 inhibition (PI) for the individual growth curves (see Tables S1, S2, and S3). The equation 607 used to calculate the percent inhibition of growth is shown in equation 6 below, from 608 (Rajnovic et al., 2019) which is reformatted in equation 7. For a full discussion of this 609 method (see Stacy and Ceballos, 2020). 610 15 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.30.017236; this version posted April 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

611 �� = (equation 6) 612 613 �� = 1 − (equation 7) 614 615 The left and right bounds of the integration process are key parameters affecting the area 616 under the curve metric. For these viral growth curves, the point of first difference is at time 617 t = 0 when virus is added. The upper bound of the growth curve was originally chosen to 618 be the intercept of the line produced by the maximum growth rate and the fitted asymptotic 619 value. This method was opted against because it does not include the transition period 620 between the exponential growth phase and the stationary phase of growth, an area of the 621 growth curve which we considered to be worthy of inclusion. Datapoints at and beyond 622 stationary phase were excluded from AUC analysis because they disproportionately value 623 the asymptotic (A) parameter, which undermines the robustness of the AUC value as a 624 unifying metric of growth curve changes. 625 626 Virus replication data, for SSV8, was fit to an exponential growth model both based on 627 peak values and AUC calculations (with similar patterns emerging with either approach): 628 629 �(�) = ��(/) (equation 3) 630 631 632 Parameter A is the initial population prior to infection and 1/τ is the growth rate constant. 633 634 For SSV9-infected cultures, average OD-based growth was fit to a sinusoidal function: 635 636 �(�) = ��(/) ∙ sin (��) (equation 4) 637 638 639 Term Ae-t/τ represents the exponential decay of the oscillation amplitude whereby A is the 640 driving amplitude and 1/τ is a decay rate constant. In the second term, ω is the frequency 641 variable. The average growth curve of SSV9-infected cultures was fit to a damped 642 sinusoidal function simply because this function best described the cyclical bursts in 643 growth that became increasing smaller in terms of amplitude and which were each followed 644 by rapid declines in host population (as determined by optical density readings). 645 However, for SSV9 replication, a Gaussian function was used to fit the dataset. 646 ( ) 647 �(�) = �� (equation 5) 648 649 Parameter A represents the amplitude of the peak function; tc is the time at which the center 650 of the peak occurs; and, w represents the width of the peak function at one-half the peak 651 amplitude. The “goodness-of-fit” of these data sets (assessed by adjusted r2 values) to the 652 sinusoidal and Gaussian, respectively, prompted their use. For the sinusoidal fit, the 653 adjusted r2 value was 0.90 (see Fig. 7B). For the Gaussian fit to the SSV9 virus replication 654 data, adjusted r2 = 0.94. Note that r2 values typically were between 0.90-0.99, except for 655 fits to the SSV9-infected host growth curves, which failed or had r2 < 0.25. 656

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657 qPCR for determination of SSV9 genome copy number. SYBR green-based qPCR was 658 used to quantify the number of SSV9 genomes for inocula and end-point infection. 659 Two PCR primers were employed: SSV9F (5'-GTGAAGCGACCAACATAGGTGCAA-3') 660 and SSV9R (5'-GTTGCGTTTGTACCGGTTACGCTA-3') - targeting the single-copy gene 661 vp1, which encodes for a major SSV structural capsid protein (Bautista, 2015). 662 Standard curves were generated with 10-fold serial dilutions (108 to 100 copies) of 663 the vp1 gene fragment (138-bp) cloned into a TOPO TA pCR2.1 plasmid (Invitrogen). 664 Copy number in each standard was calculated using formula: 665 ∙ µ 666 �� = (equation 6) [ ()] ∙ ∙ (/) 667 668 669 where, 660 g/mol is the molecular weight of one base pair, NA is Avogadro’s number, 670 and 1x109 is used to convert units to nanograms. Each qPCR reaction was performed 671 on a Rotor-Gene Q (Qiagen, Inc.; Maryland, USA) and consisted of 1X Green/ROX qPCR 672 Master Mix (Fermentas, Inc.; Ontario, Canada), 3 pmol of each primer, 1 uL of DNA 673 template, and nuclease-free water up to a final volume of 20 uL. Three technical 674 replicates were performed of each standard and samples. The qPCR cycle parameters 675 were as follows: 98°C for 2 minutes, followed by 40 cycles of 98°C for 5 s and 20s at 676 60°C. A melt curve analysis was performed after each run from 65 to 95°C in 0.5°C 677 increments at 2 s intervals, to ensure specific amplification of a single target, and no 678 primer dimer formation. The qPCR amplicons were resolved in 2% agarose gels to 679 assess amplicon sizes for target-specific amplification. Reaction mixtures with sterile 680 water (no-template DNA) or pCR2.1 DNA without insert served as a negative controls 681 to control for false positives. Assays showed amplification efficiencies of 100% ± 10% 682 (i.e., with a slope between −3.6 and −3.1), consistency across replicate reactions, and 683 linear standard curves (R2 > 0.970). The absolute quantification method was used to 684 calculate the number of viral genomes per mL of culture. 685 686 AUTHOR CONTRIBUTIONS 687 688 RMC and KS conceptualized the study. RMC performed liquid culture and plate assays. 689 RMC did virus titering using ESI/MS with instrumentation at BVS, Inc. (Hamilton, MT). 690 CD performed plate assays and conducted the SSV thermal stability and adsorption studies. 691 CD also did virus titering using serial dilution plaque assays. CLS and RMC fit data to

692 mathematical models and determined Nasymptote, µmax, and AUC for all liquid culture assays. 693 RMC and CD produced TEM and SEM images. EP did qPCR-based virus titers. 694 695 FUNDING 696 697 Distinct components of this project were supported in part by each of the following sources: 698 NASA EPSCoR award no. NNX07AT63A (EPSCoR PD-DesJardins; SciPI-Ceballos); 699 U.S. NSF RIG award no. 0803199 (PI-Ceballos); U.S. NSF MCB award no. 1818346 (PI- 700 Ceballos); U.S. NSF MCB award no. 0702020 (PI-Stedman).

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701 ACKNOWLEDGEMENTS 702 703 The authors thank: UA students Kyle Hanson and Blythe Bunkers for assisting with 704 infection assays; David Wick (BVS, Inc.) for technical support with the ESI/MS titering; 705 and, Dr. Jonathan Trent and Dr. Mark Young for providing several strains of Sulfolobales 706 derived from different geothermal regions worldwide for comparative analyses. 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747

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748 749 SUPPLEMENTARY MATERIAL 750 751 752 Virus AUC Virus Count Virus Titer Strain Calculated Estimated Calculated SSV1 2141.25 180 VP/nL 1.80 x 108 VP/mL SSV8 3217.85 268 VP/nL 2.68 x 108 VP/mL ) SSV9 5560.70 463 VP/nL 4.63 x 108 VP/mL

600nm 120 nL of 1:10 dilution of virus stock aerosolized over a 3 min. ESI/MS run Optical Density (OD Density Optical

Hours Post-Inoculation (HPI) 753 754 755 FIGURE S1 - SSV Titers via Electrospray Ionization Mass Spectrometry (ESI/MS). Concentrated 756 viral suspension is diluted 1:10 and mixed with ionization solution (see Methods section). SSV particle 757 count in 120nL of suspension is monitored by a particle detector for 3 min. The area-under-the-curve 758 (AUC) provides an estimate of titers. Each SSV has a characteristic spectrum. SSV1 (blue) is generally a single broad peak. SSV8 (green) exhibits a large single peak with a shoulder. SSV9 (red) typically 759 presents as a double-peak. Since ESI/MS measures are based on mass:charge ratios of spherical particles 760 and SSVs are fusiform (quasi-elliptical) shoulders and double peaks may be due to different orientations 761 during between aerosolization and triggering the detector. Alternatively, morphologically different 762 populations may be present, including a sub-population of defective virus-like particles such as those 763 that have lost their hydrophobic tails, which generate a shoulder or second peak. 764 765 766 767 768 769 770 771 772 773

S. solfataricus growth curves 0.9 0.8 0.7 0.6 0.5 0.4 GƟ OD 600 0.3 GƟ + SSVK‐1 0.2 GƟ + SSV1 0.1 0 0 20 40 60 80 100 120

Hours Post Infection

Figure 4: Infected Sulfolobus solfataricus growth curves. Optical densities (OD600) of S. solfataricus GƟ cultures infected with SSV1 (green triangles), SSVK-1 (red triangles), and bioRxiv preprint doi: https://doi.org/10.1101/2020.03.30.017236; this version posted April 1, 2020. The copyright holder for this preprint (which uninfectedwas not certified by(blue peer review) squares). is the author/funder. Initial All rights time reserved. points No reuse are allowed 4 withouthours permission. after infection, after cells were washed.

774

775 776 q 777 FIGURE S2 - Debris in SSV9-infected Small-Scale Cultures. (A) SSV9-infected Sulfolobus strain G Figureshows significantly 5: Sulfolobus more cell debris solfataricus at 22HPI than (B) SSVGƟ-infected cultures. Gq at 22 HPISamples and (C) uninfected of one -step growth curve cultures 39 778 Gq uninfected control. Both infections were conducted at an MOI=3 where virus titer was determined by 779 hoursplate-based post serial infection. dilution plaque -A)like “halo”S.solfataricus assays (units = haloGƟ-forming infected units/mL with or hfu/mL) SSVK. Cell- 1, with apparent cell debris. B) 780 debris is another line of evidence suggesting SSV9-induced host cell lysis. 781 S.solfataricus GƟ infected with SSV1. C) S.solfataricus GƟ without the addition of virus. The 782 783 dark region at the bottom of all three tubes is a reflection. Contrast was automatically adjusted in 784 Photoshop CS4 and all three tubes are from the same photograph. Tubes have not been 785 786 centrifuged or otherwise treated. 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 17 806 807 808 809 810

20 formation were observed. Thus, halo formation occurred independently of the amount of time formation were observed. Thus, halo formation occurred independently of the amount of time plates were left at room temperature. plates were left at room temperature.

SSV infectivity at 75ºC 811 SSV infectivity at 75ºC 1.0E+06 812 813 1.0E+06 1.0E+05 814 1.0E+05 1.0E+04 815 fu/mL) 816 1.0E+04 817 fu/mL)

er h 1.0E+03 818 SSV1 er h 1.0E+03 SSV1 1.0E+02 819 SSV1 820 1.0E+02 SSSSV9VK‐1 Virus tit 1.0E+01 821 SSVK‐1 Virus tit 822 SSV Titers (hfu/mL) 1.0E+01 1.0E+00 823 1.0E+00 824 0 5 10 15 20 25 30 825 0 5 10 15 20 25 30 826 Time (hours) 827 Time (hours) 828 829 5 Figure 2: SSV stability830 at 75°C. Purified SSV1 and SSVK-1 stocks in YS-medium at 5*10 FIGURE S3 - SSV Viability After Exposure to Culture Environment. At 75°C (pH 3.2) SSV9 (red) retains 5 hfu/mL were incubatedFigure831 2 at: SSV75°Cviability stability forfor up theto 25 hours;indicated at 75°C. while titers times.Purified of SSV1 At(blue) SSV1 2, decrease 6, 18, and rapidly 24 SSVK after and 6 hr 26- in1 the stockshours same high samplesin temperature YS-medium at 5*10 832 acid conditions. Plate-based serial dilution plaque-like “halo” assays (hfu/mL) were used to determine titers were taken and hfuvirus/mL titer833 were determinedfor eachincubated time point viafor at SSV9 halo75°C and SSV1.assay. for SSV9the Both indicatedvirus particlesSSV- (red) 1times. (blue have greater Atdiamonds) 2,stability 6, 18,in the and 24high andtemperature SSV 26-K1 hours samples (red squares) exhibitedwere taken834 initial andand acid drops virus environment. intiter titer, determined though the via SSV1 halo sampleassay. Both had noSSV detectable-1 (blue diamonds)virus and SSV-K1 (red squares)835 exhibited initial drops in titer, though the SSV1 sample had no detectable virus (<500 hfu/mL) after 6836 hours, while SSVK-1 retained infectivity after 26 hours of incubation. (<500 837hfu/mL) after 6 hours, while SSVK-1 retained infectivity after 26 hours of incubation. 838 839 840 841 Adsorption timing: 842 843 Adsorption844 timing: Accurate measurement845 of virus production by newly infected cells requires washing 846Accurate measurement of virus production by newly infected cells requires washing 847 away excess viruses used848 in the initial infection, following their adsorption to cells. To ensure away excess849 viruses used in the initial infection, following their adsorption to cells. To ensure 850 that cells were not washed851 prior to infection taking place, adsorption time was determined that cells852 were not washed prior to infection taking place, adsorption time was determined 853 indirectly by loss of virus854 titer in the presence of Sulfolobus solfataricus GƟ cells at an MOI of indirectly855 by loss of virus titer in the presence of Sulfolobus solfataricus GƟ cells at an MOI of 0.01, (100 fold excess856 cells). Because virus titers of concentrated SSV1 and SSVK-1 stocks 0.01, (100 fold excess cells). Because virus titers of concentrated SSV1 and SSVK-1 stocks decreased significantly at 75ºC in the absence of cells (Figure21 2), this assay was performed at decreased significantly at 75ºC in the absence of cells (Figure 2), this assay was performed at 23°C to analyze the loss in virus titer due to the presence of cells independent from the loss in 23°C to analyze the loss in virus titer due to the presence of cells independent from the loss in

14 14 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.30.017236; this version posted April 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

857

858 859 860 FIGURE S4. SSV9 Genome Abundance via Quantitative Polymerase Chain Reaction (qPCR). 861 Viral genome counts were measured using qPCR For S150-SSV9 and S147-SSV9 challenges. Inoculation of S150 and S147 cell suspensions with SSV9 at an MOI = 0.1 (dark grey bars) from a 862 10 SSV stock titered at 1.03x10 vg/mL (black bar) was followed by incubation for 72 hours at 78°C 863 (90 RPM shaking). At end-point 72 HPI, titers were determined by qPCR from culture supernatant. 864 The presence of SSV9 genomes per mL in S150 culture dropped five orders of magnitude to within 865 the lowest detectable limits of the system (medium grey bar), which indicates no productive infection. 866 SSV9 genomes in S147 culture increased by two orders of magnitude (light grey bar), indicating that 867 S147 is permissive to SSV9 replication. 868 869 870 871 872 873 874 875 876 877

22 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.30.017236; this version posted April 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

878 879 Table S1 – Percent Inhibition Calculations for SSV-Gq Infection: Figure 4.

Trial AUCRiemann AUCGompertz PI (%) R2Gompertz

Gq-CTLUNINFECTED 61.851 62.15 0 0.975 Gq-SSV1 56.454 56.83 8.6 0.988 Gq-SSV2* 43.017 43.19 30.5 0.968 Gq-SSV3* 42.972 43.42 30.1 0.951 Gq-SSV8 44.274 44.47 28.4 0.978 Gq-SSV10 51.57 51.97 16.4 0.9242 880 881 * AUC values for SSV2 and SSV3 are underestimated since the curves had not reached stationary 882 phase by the end of the trial time period; therefore, direct comparisons of PI are not appropriate. 883 SSV2 and SSV3 show a delay in reaching stationary phase (i.e., Nasymptote)when compared to SSV1 884 and SSV8. Extrapolated values predict that PI for SSV2 and SSV3 would be approximately equal 885 to that shown for SSV1. 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920

23 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.30.017236; this version posted April 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

921 922 923 Table S2 - Percent Inhibition for SSV Infections on Allopatric Hosts: Figure 5.

Trial AUC PI (%) R2Gompertz SEAUC (±)

S200-CTLAVG 113.4 0 0.978 0.792

S200-SSV1AVG 107.5 5.3 0.986 4.104

S200-SSV8AVG 75.0 33.9 0.965 0.691

S200-SSV9AVG 31.4 72.4 0.301 1.332

S444-CTLAVG 70.3 0 0.976 1.778

S444-SSV1AVG 59.6 15.1 0.980 1.070

S444-SSV8AVG 51.8 26.3 0.970 0.357

S444-SSV9AVG 24.2 65.6 0.479 1.172

S437-CTLAVG 102.6 0 0.962 0.221

S437-SSV1AVG 99.0 3.5 0.981 1.603

S437-SSV8AVG 69.9 31.8 0.969 6.272

S437-SSV9AVG 31.6 69.2 0.531 4.728 Coefficients of Determination (R2) less than 0.8 (in bold) are considered “failed” fits to the Gompertz model. For strain descriptions and references see main text. 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 24 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.30.017236; this version posted April 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

951 952 953 Table S3 - Percent Inhibition for SSV9 Challenges on Sympatric Sulfolobales: Figure 8.

Trial AUC PI (%) R2Gompertz SEAUC (±)

S150-CTL1 54.1 -1.5 0.935

S150-CTL2 51.5 3.5 0.857

S150-CTL3 54.4 -1.9 0.924

S150-CTLAVG 53.3 0 0.920 0.92

S150-SSV91 53.3 0.0 0.931

S150-SSV92 54.5 -2.2 0.913

S150-SSV93 55.8 -4.7 0.941

S150-SSV9AVG 54.5 -2.3 0.942 0.72

S147-CTL1 42.7 1.0 0.917

S147-CTL2 39.9 7.3 0.946

S147-CTL3 46.7 -8.3 0.955

S147-CTLAVG 43.1 0 0.967 1.97

S147-SSV91 31.8 26.3 0.912

S147-SSV92 31.4 27.2 0.960

S147-SSV93 30.4 29.6 0.843

S147-SSV9AVG 31.2 27.7 0.934 0.41 For strain descriptions and references see main text. 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973

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