Specialization to cell-free or cell-associated spread by BAC-cloned HCMV strains not determined by the UL128-131 and RL13 loci

A widely held view is that clinical isolates of human cytomegalovirus (HCMV) are cell-associated and that mutations affecting the UL128-131 and RL13 loci arise during subsequent passage in culture and lead to the appearance of a cell-free spread phenotype. To distinguish the factors influencing cell-associated and cell-free spread, we analyzed the spread characteristics of three HCMV BAC-clones; Merlin (ME), which expresses high levels of UL128-131 and harbors a frameshift mutation within RL13, and TB40/e (TB) and TR, which are both low in UL128-131 and intact at RL13. Quantitation of the number of newly infected cells over 12 days by flow cytometry revealed remarkably similar spread efficiencies in fibroblasts among strains. However, comparing the inhibition of spread by neutralizing antibodies and the quantities and infectivity of progeny virus indicated that TB and TR spread was predominately cell-free, whereas spread of ME was predominantly cell-associated. While transcriptional repression of UL128-131 greatly enhanced cell-free spread by ME, the efficiency of the cell-associated mode was not affected. Spread in epithelial cultures was highly cell-associated for all strains, and ME was the most efficient. Repression of UL128-131 reduced the efficiency of ME spread in epithelial cells, but did not affect the predominate mode of spread. Spread in RL13-expressing cells was comparably reduced for all strains, and more pronounced in fibroblasts than in epithelial cells. RL13 effects could not be clearly explained by changes in production, release, or infectivity of progeny virus, and there were no changes to the proclivity of strains for cell-free or cell-associated spread. In sum, the specialization of HCMV strains to cell-free spread is linked to the quantity and infectivity of cell-free progeny, which can be influenced by UL128-131 levels, but the cell-associated specialist phenotype is likely determined by factors beyond the UL128-131 or RL13 loci. AUTHOR SUMMARY Experimental distinctions between cell-free and cell-associated modes of spread for HCMV have been largely relativistic. When cell-free spread is inhibited by neutralizing antibodies, or if particular strain of virus is poor at cell-free spread, then the observed spread may be simply defined as “cell-associated”. However, such a view does not easily lend towards analysis of the efficiency of cell-associated spread or the factors involved. In our study, we measured the kinetics of HCMV cell-free and cell-associated spread as independent processes and show evidence that HCMV strains can be highly specialized to one or the other mode of spread. The genetic factors that determine specialization for mode of spread are unclear, but given the genetic diversity of HCMV circulating in human populations, it seems likely that both modes of are represented. The efficacy of intervention approaches is likely affected by the mode of spread. For example, neutralizing antibodies are less effective to limit cell-associated spread. Our results provide a conceptual approach to evaluating intervention approaches such as neutralizing antibodies raised by vaccine candidates and drug compounds that target viral replication processes for their ability to limit cell-free or cell-associated modes of spread as independent processes.


INTRODUCTION
virus was complicated by the fact that a large number of intracellular viral genomes likely remain in the by far the least infectious ( Fig 3D).

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In epithelial cells, the release of cell-free virus was notably different among strains (Figs 4A and C).

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Note that while TB released 6-fold fewer cell-free progeny than ME, the cell-free TB progeny were 5-fold more 157 infectious than the ME progeny (compare Figs 4 A and B). This indicates that TB and ME generate 158 comparable amounts of total infectivity into epithelial cell culture supernatants. In contrast, TR produced far 159 less total cell-free infectivity in these cultures since the low quantity of progeny released was not compensated 160 by better infectivity. The accumulation of epithelial cell-associated progeny was comparable among strains 161 and the cell-associated infectivity was within the range of 15,000-45,000 genomes/IU for all strains ( Fig 4C   162 and D.).

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To further evaluate the contribution of cell-free and cell-associated spread mechanisms, neutralizing 164 antibodies were used with the rationale that they inhibit cell-free spread more potently than cell-associated 165 spread. Neutralizing antibodies chosen for these experiments were mAb 14-4b, which likely targets a 166 discontinuous epitope at the membrane proximal region of gH (51, 52), as well as a mixture of rabbit anti-167 peptide sera that target UL130 and UL131 (17). The relative potency of these antibodies to neutralize cell-168 free TB, TR, and ME was verified in neutralization experiments (Fig. S1). Spread rates of each strain in 169 either fibroblast or epithelial cell cultures were then determined in the presence or absence of anti-gH mAb 14-170 4b or anti-UL130/131 sera at concentrations sufficient for complete neutralization of cell-free virions. To 171 demonstrate that the observed spread required production of progeny viral genomes and was not the result of transfer of cytoplasmic contents from infected to uninfected cells, spread in the presence of ganciclovir was had no effect on the spread rates of any of the three strains in fibroblasts (Fig 5, A-C). The residual spread of 179 all strains in the presence of neutralizing antibodies was greater than with ganciclovir treatment, even for TR, 180 which is known to harbor resistance mutations in the UL97 kinase (53). These results support the hypothesis 181 that spread of TB in fibroblasts predominately involved cell-free virions that were sensitive to neutralizing 182 antibodies, whereas ME spread predominately in a manner not sensitive to antibody neutralization (i.e., not by

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In epithelial cell cultures, the presence of anti-gH 14-4b had no effect on the spread rates for TB, TR, 189 or ME, and anti-UL130/131 had a marginal effect, with a 30% reduction for TR being the largest (Fig 5D-F). If

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(TetO) sequences in the promoter of UL131. We previously showed that replication of this recombinant ME in 212 fibroblasts expressing the tetracycline-repressor (TetR) protein produced extracellular virus with amounts of 213 gH/gL/UL128-131 at or below that of TB and TR, marginally higher amounts of gH/gL/gO, and these changes 214 in glycoproteins greatly improved cell-free infectivity (27). In order to test the role of high UL128-131 215 expression for ME spread, experiments were conduced using these TetR-expressing fibroblasts (HFFFtet), as 216 well as a newly generated TetR-expressing ARPE19 epithelial cell line (ARPEtet). Both HFFFtet and ARPEtet 217 showed efficient repression of a TetO-regulated luciferase reporter (Fig. 6A). Consistent with our previous 218 characterizations, ME virions produced in HFFFtet cells contained drastically reduced amounts of 219 gH/gL/UL128-131, increased gH/gL/gO, and infectivity was dramatically improved (Fig 6B and C). Similarly, 220 ME virions produced in ARPEtet cells contained dramatically reduced amounts of gH/gL/UL128-131 221 compared to virions from control ARPE cells, but did not contain increased gH/gL/gO, or display enhanced 222 infectivity as was observed for fibroblasts-derived virus (Fig 6B). Note that even without repression of 223 gH/gL/UL128-131, the specific infectivity of epi-ME on epithelial cells was dramatically better than fib-ME on 224 fibroblasts ( Fig 6C).

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In fibroblasts, gH/gL/UL128-131 repression did not influence spread rates or the sensitivity of spread to 226 neutralizing antibodies ( Fig 7A). Thus, the efficient cell-associated spread of ME in fibroblasts does not 227 require high levels of gH/gL/UL128-131. In epithelial cells, repression of gH/gL/UL128-131 resulted in a 228 modest decrease in average spread rate (from 0.38 to 0.29), and small increase in the sensitivity of spread to blocking of the cell-associated spread by these antibodies. In any case, ME spread in ARPEtet cells was still 235 highly resistant to antibodies as compared to the spread of TB in fibroblasts (compare 30% antibody resistant 236 spread for TB in fibroblasts; Fig 5A, to 70% resistant for ME in ARPEtet; Fig 7B). Overall, these results 237 indicated that while reduction of gH/gL/UL128-131 may enhance cell-free modes of spread, at least on 238 fibroblasts, high expression levels of the gH/gL/UL128-131 could not explain the highly specialized cell-239 associated spread of ME.

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Spread inhibition due to RL13 expression is more pronounced in fibroblasts than in epithelial 241 cells, and influences both cell-free and cell-associated modes of spread. The RL13 ORF encodes an 242 envelope glycoprotein that has been described as an inhibitor of HCMV replication in culture and mutations in 243 RL13 have been documented during cell-free passage in fibroblasts and epithelial cells (40, 54, 55). While 244 the ME recombinant used in the present studies harbors such a RL13 mutation, the RL13 expression status of 245 the TB and TR BAC-clones is unclear. Both TB and TR have intact RL13 ORFs confirmed by DNA 246 sequencing, but lack of quality antibodies makes protein detection difficult. To compare the effect of RL13 on 247 the spread of all three strains, and to avoid potential selection of RL13 mutants during spread experiments, 248 fibroblast and epithelial cell lines that express RL13 were engineered. Immunoblot analysis demonstrated that 249 these cell lines expressed of both mature and immature proteoforms, and flow cytometry confirmed similar 250 expression levels ( Fig S2). The effects of RL13 expression on spread rates, and the quantity and infectivity of 251 cell-free and cell-associated progeny virus were then analyzed (Fig 8).

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In fibroblasts, RL13 expression resulted in a comparable 40-50% reduction in spread rate for all three 253 strains ( Fig 8A). Inhibition of spread by neutralizing antibodies was not affected for TB or TR, but spread of 254 ME in RL13 expressing cells was approximately 50% more sensitive than in control cells (Fig 8B). These 255 observations could not be universally explained by effects on progeny virus production since effects of RL13 statistically significant change in progeny infectivity due to RL13 expression was observed for cell-free TB, 258 which was 6-fold less infectious than virus produced from control fibroblasts (Fig. 8D).
antibody inhibition, whereas TR and ME were less affected ( Fig 8F). As in fibroblasts, these effects were not 262 readily attributable to the quantity or infectivity of progeny since 1) there were no significant changes to the 263 quantity of cell-associated or cell-free progeny virus for any of the three strains (Fig 8G), and 2) the only 264 notable effect on progeny infectivity was in the case of cell-free ME, which was 120-fold less infectious when 265 produced in RL13-expressing epithelial cells as compared to control cells (Fig. 8H).

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To determine if the observed reduction in infectivity of cell-free ME produced in RL13 expressing 267 epithelial cells was related changes in the virion content of gH/gL complexes or gB, equal quantities of 268 fibroblast-or epithelial-derived extracellular virions of ME were subjected to non-reducing SDS-PAGE and 269 immunoblot analysis with antibodies specific for gL to detect gH/gL complexes, or with anti-gB monoclonal 270 antibodies that react with both the full length 165-170 kDa form and the 55 kDa portion of the furin-cleaved 271 form of gB (56-58) (Fig 9). Fib-ME virions contained gH/gL primarily in the form of gH/gL/UL128-131, and 272 most of the gB was in the furin-cleaved form. RL13 expression in fibroblasts seemed to have little effect on 273 either gH/gL complexes or gB. However, there were notable differences in epithelial cells. First, epi-ME 274 virions contained greater amounts of gH/gL/UL128-131 than fib-ME, and this was somewhat reduced by RL13 275 expression. Secondly, epi-ME had more uncleaved gB, whereas ME virions produced by RL13 expressing 276 epithelial cells had less overall gB, and it was mostly furin-cleaved. Together, these results indicated that the 277 processing and incorporation of gH/gL complexes and gB into cell-free progeny can be influenced by the cell 278 type, and in epithelial cells, this may be further affected by RL13 status. While the effects on gB seem to 279 correlate with the observed reduction in ME infectivity in RL13 epithelial cells, a strong causal connection 280 would be a premature conclusion.

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In sum, these results indicate that 1) RL13 expression tempers the spread of HCMV in fibroblasts by 282 either the cell-free mode exemplified by TB, or the cell-associated mode exemplified by ME, 2) The effect of gB. the mechanistic differences, and the genetic loci involved in these modes of spread remain to be understood. passage clinical isolates spread more rapidly than others (61). They interpreted slow kinetics to represent a 303 cell-associated-only phenotype and fast kinetics to represent a phenotype of both cell-associated and cell-free 304 spread. Indeed, the faster spreading isolates were more sensitive to the presence of neutralizing antibodies.

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Together, these and other studies suggest considerable variation in the modes of spread exhibited by 306 genetically distinct HCMV, but the exact genetic variations that determine these phenotypes remain unclear.

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Here we applied a flow cytometry-based approach to compare the spread characteristics of three 308 commonly studied and genetically distinct BAC clones of HCMV, TB, TR and ME in both fibroblast and 309 epithelial cell cultures. In addition to measuring the rates of spread in the presence or absence of neutralizing 310 antibodies, we also characterized the quantity and infectivity of cell-free and cell-associated progeny infectivity. In both cell types, ME was far more efficient at cell-associated spread than TB or TR, but did not 318 produce more cell-associated infectivity as measured by titration of sonicated infected cells. This suggests 319 that the efficient cell-associated spread mechanism of ME depends on the intact cell monolayer. Moreover, it 320 seems logical that the poor infectivity of ME virions would be highly detrimental to efficient cell-free spread, 321 whereas cell-associated mechanisms might be less sensitive to poor infectivity. On the other hand, it seems 322 unlikely that the log-folds better infectivity of TB and TR virions would be detrimental to the efficiency of cell-323 associated spread by these strains (Fig 10). Thus, it seems that ME has a specific mechanism(s) to enhance 324 cell-associated spread that does not depend on production of highly infectious virions. The mechanisms 325 driving the highly efficient cell-associated spread of ME remain unclear, but have been suggested to involve 326 the functions of gH/gL/UL128-131 and RL13 (24, 40, 42, 62).

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Our finding that the level of gH/gL/UL128-131 expression did not affect the spread rate of ME in 328 fibroblasts conflicts with Stanton et al., which showed UL128-null ME plaques significantly larger than UL128-  Thus, the increase in 21 day plaque size of a UL128-null ME documented by Stanton is consistent with 337 enhanced cell-free infectivity, but our analysis of spread rate over 12 days indicates this does not come at the 338 expense of a distinct and comparably efficient mode of cell-associated spread. This interpretation is 339 supported by the observation that the 12 day spread rate was highly resistant to neutralizing antibodies the 12 day spread rate for ME (Fig 8). This was consistent with reduced plaques sizes of UL128-null ME 344 documented by Murrell et al., and likely reflects the important role of gH/gL/UL128-UL131 in epithelial cells 345 (38). However, as observed in fibroblasts, spread in epithelial cells was still highly resistant to neutralizing 346 antibodies when gH/gL/UL128-131 was repressed. This was different than Murrell et al. who concluded that 347 spread of ME in epithelial cells was more sensitive to antibodies when gH/gL/UL128-131 was reduced (42).

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Again the discrepancy likely reflects that analysis of 21-day plaque size likely accentuates the effects of cell-349 free infectivity and masks the contribution of cell-associated spread. Thus, by measuring average spread 350 rates over 12 days, we find that gH/gL/UL128-131 levels do not determine the cell-free versus cell-associated 351 spread phenotype for ME on either fibroblasts or epithelial cells.

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The RL13 glycoprotein was another candidate for a factor influencing the cell-free and cell-associated

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RL13 expression reduced the release of cell-free progeny, but this effect was less pronounced for TR or ME 359 on fibroblasts, and there was no effect on cell-free progeny release in epithelial cells for any strain.

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Interestingly, RL13 expression dramatically reduced the infectivity of cell-free virions released by ME from 361 epithelial cells, and this was consistent with results of Stanton et al. showing low cell-free titers for RL13-intact 362 ME in epithelial cell cultures compared to RL13 mutant ME (40). The physiological basis of this effect on ME 363 infectivity remains unclear, but it seemed to correlate with differences in gH/gL complex and gB incorporation.
neutralizing antibodies in RL13-expressing cells was either unaffected or marginally increased for all three 370 strains. Thus, RL13 seems to dampen one or more aspects of HCMV replication that can manifest through 371 either mode of spread. The specific effects of RL13 may vary depending on other physiologic differences 372 between strains and cell types.
a phenomenon that has been universally ascribed to HCMV, despite clear strain variably. While ME is highly similar to ME in their proclivity for cell-associated spread, (note that ME was also isolated from a urine sample 390 (10)), it would seem logical that neutralizing antibodies would reduce the selective advantage of cell-free 391 infectivity conferred by UL128-131 mutation. The preservation of RL13 is less intuitive since RL13 seems to

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The accumulation at day 7 from nHDFs and ARPEs is shown in Fig 3 and 4, respectfully.                                         000 or the cell-associated (4b) route may be mediated by either direct fusion between the virion envelope and 001 plasma membrane, or by fusion from within an endosome. 5. Alternatively, some studies have suggested that 002 subviral components may be transferred directly to adjacent cells through transient pores or other intercellular 003 connection mechanisms, such as tunneling nanaotubes (45, 76, 77). Overall, specific infectivity of virions 004 might have a dramatic effect on the efficiency of the cell-free route (2a-4a) where the virus is subject to 005 physical limitations related to diffusion and to inactivation by neutralizing antibodies, complement components, 006 and other host defenses. In contrast, the efficiency of cell-associated routes (2b-4b, and 5) might be more 007 influenced by mechanisms of directed trafficking and intercellular connectivity.