A New Approach to Assessing HSV-1 Recombination during Intercellular Spread

Abstract

The neuroinvasive Herpes simplex virus type 1 (HSV-1) utilizes intergenomic recombination in order to diversify viral populations. Research efforts to assess HSV-1 recombination are often complicated by the use of attenuating mutations, which differentiate viral progeny but unduly influence the replication and spread. In this work, we generated viruses with markers that allowed for classification of viral progeny with limited attenuation of viral replication. We isolated viruses, harboring either a cyan (C) or yellow (Y) fluorescent protein (FP) expression cassette inserted in two different locations within the viral genome, in order to visually quantify the recombinant progeny based on plaque fluorescence. We found that the FP marked genomes had a limited negative affect on the viral replication and production of progeny virions. A co-infection of the two viruses resulted in recombinant progeny that was dependent on the multiplicity of infection and independent of the time post infection, at a rate that was similar to previous reports. The sequential passage of mixed viral populations revealed a limited change in the distribution of the parental and recombinant progeny. Interestingly, the neuroinvasive spread within neuronal cultures and an in vivo mouse model, revealed large, random shifts in the parental and recombinant distributions in viral populations. In conclusion, our approach highlights the utility of FP expressing viruses in order to provide new insights into mechanisms of HSV-1 recombination.

Keywords: HSV-1; alphaherpesvirus; cell–cell spread; fluorescent protein; intravitreal injection; neuroinvasion; neuron culture; recombination.

Figure 1

Characterization of the genetically tagged Herpes simplex virus (HSV) genomes. (A) Schematic representation of the fluorescent protein expression cassette location on the HSV-1 genome. Boxes represent large terminal and internal repeat sequences. Expression cassettes for yellow fluorescent protein (YFP)- and cyan fluorescent protein (CFP)-NLS proteins have been inserted in the UL36/37 and US1/2 loci, respectively. The genetic insertions result in different fluorescent expression phenotypes of the resulting viral plaques (images in panels below, scale bar = 100 μm). Parental infections result in YFP and CFP positive plaques. Upon co-infection, the genomes can recombine, producing progeny containing both of the genetic cassettes. Those plaques will either express both of the fluorescent proteins (Dual FPs) or the lack fluorescent protein expression (No-FP); (B) single step replication plots of parent viral isolates; HSV YFP_UL, HSV CFP_US, and HSV-1 strain 17. The proportion of the viral progenies were plotted following the (C) direct competition assay between parent viruses and the (D) indirect competition assay against HSV-1 strain 17. The indicated FP expressing HSV virus or HSV-1 strain 17 were co-infected at an MOI of 10 for each virus. The total infected cells were harvested at 8 hpi. The harvested virus was plated at a limiting dilution on the cell monolayers and plaques and was scored based on the fluorescent protein expression. A statistical t-test comparison of the direct and indirect competition assays revealed significant differences between the wild-type and FP expressing viruses.

Figure 2

Effects of time and MOI on recombinant progeny output. (A) The effect of time during replication was assessed by counting the distribution of the fluorescent plaques during a time course of viral infection. Cells were infected at an MOI of 10 and subsequently harvested at 0, 3, 6, 9, 12, and 24 h post-infection. Infected cell lysates were plated at a limiting dilution and the resulting progenies were scored for the number of fluorescent plaques from each of the four genome types; (B) the effect of MOI on the total recombinant progeny production was assessed by differential inoculation. Vero cells were inoculated at an MOI of 0.1, 1, 5, 10, or 50 of the HSV YFP and HSV CFP viruses and the viral progeny was harvest at 8 hpi. As MOI increases, the frequency of the recombinant progeny increases with logarithmic progression. When the data are expressed as a plot of % recombinant progeny versus the log₁₀ value of the MOI, the resulting linear slope is achieved with a logarithmic regression. Presented is a representative experiment involving replicates of three at each MOI that was evaluated, with standard deviation plotted between replicates.

Figure 3

Recombinant progeny after multiple cycles of infection. A mixed population of HSV-1 viruses were generated following co-infection (inoculum) and progeny viruses were sequentially passaged three times. The inoculum and each subsequent passage were analyzed for content of the individual FP expression. Each plaque phenotype is plotted as a percent of the total plaque distribution. Data represents the average of the triplicate samples, with error bars as the standard deviation between replicates. Significant changes were only observed in the No-FP populations, as evaluated by one-way ANOVA.

Figure 4

Effect of transneuronal spread on populations of recombinant viruses. (A) A schematic representation of the compartmentalized neuronal culture system. Briefly, a Teflon ring is attached to the culture surface. Dissociated superior cervical ganglions (SCG) neurons are plated in the left cell body compartment. Those cell bodies extend axon projections under the two internal walls, resulting in isolated axon termini in the Axon Compartment. Prior to infection, detector cells (not depicted in diagram) are plated in the axon compartment to amplify infectious progeny that transmit via axon-to-cell spread; (B) after 48 h of the cell body compartment inoculation, both compartments were harvested and plated at limiting dilution. The plaques developing from the viral progeny were analyzed for the distribution of FP expression in the cell body and axon compartments. * denotes p < 0.05, ** denotes p < 0.005 as identified through pairwise t-test comparison.

Figure 5

Effect of neuroinvasive spread on populations of viruses in vivo. (A) A schematic of the murine eye model for neuroinvasive spread; (B) frequency of parental progeny and recombinant progeny obtained from the eye, contralateral midbrain (CLMb), ipsilateral midbrain (ILMb), and hindbrain (Hb). Data presented are the summation of the two experiments for both C57Bl/6 and Balb/C mice. Each experiment contained between four and five mice; (C) distribution of FP distributions from the eye, CLMb, ILMb, and Hb are plotted. The top row shows the average distribution of plaques for the our sites in both C57Bl/6 and Balb/C mice. The lower rows are two example distributions from independent mice from each experimental group. Significant differences indicated were identified by two-way ANOVA analysis.