Entry of Herpes Simplex Virus Type 1 (HSV-1) into the Distal Axons of Trigeminal Neurons Favors the Onset of Nonproductive, Silent Infection

Following productive, lytic infection in epithelia, herpes simplex virus type 1 (HSV-1) establishes a lifelong latent infection in sensory neurons that is interrupted by episodes of reactivation. In order to better understand what triggers this lytic/latent decision in neurons, we set up an organotypic model based on chicken embryonic trigeminal ganglia explants (TGEs) in a double chamber system. Adding HSV-1 to the ganglion compartment (GC) resulted in a productive infection in the explants. By contrast, selective application of the virus to distal axons led to a largely nonproductive infection that was characterized by the poor expression of lytic genes and the presence of high levels of the 2.0-kb major latency-associated transcript (LAT) RNA. Treatment of the explants with the immediate-early (IE) gene transcriptional inducer hexamethylene bisacetamide, and simultaneous co-infection of the GC with HSV-1, herpes simplex virus type 2 (HSV-2) or pseudorabies virus (PrV) helper virus significantly enhanced the ability of HSV-1 to productively infect sensory neurons upon axonal entry. Helper-virus-induced transactivation of HSV-1 IE gene expression in axonally-infected TGEs in the absence of de novo protein synthesis was dependent on the presence of functional tegument protein VP16 in HSV-1 helper virus particles. After the establishment of a LAT-positive silent infection in TGEs, HSV-1 was refractory to transactivation by superinfection of the GC with HSV-1 but not with HSV-2 and PrV helper virus. In conclusion, the site of entry appears to be a critical determinant in the lytic/latent decision in sensory neurons. HSV-1 entry into distal axons results in an insufficient transactivation of IE gene expression and favors the establishment of a nonproductive, silent infection in trigeminal neurons

Inhibition of Superinfection and the Evolution of Viral Latency ▿

Latent viruses generally defend their host cell against superinfection by nonlatent virulent mutants that could destroy the host cell. Superinfection inhibition thus seems to be a prerequisite for the maintenance of viral latency. Yet viral latency can break down when resistance to superinfection inhibition, known as ultravirulence, occurs. To understand the evolution of viral latency, we have developed a model that analyzes the epidemiology of latent infection in the face of ultravirulence. We show that latency can be maintained when superinfection inhibition and resistance against it coevolve in an arms race, which can result in large fluctuations in virulence. An example is the coevolution of the virulence and superinfection repressor protein of phage λ (cI) and its binding target, the λ oLoR operator. We show that this repressor/operator coevolution is the driving force for the evolution of superinfection immunity groups. Beyond latent phages, we predict analogous dynamics for any latent virus that uses a single repressor for the simultaneous control of virulence and superinfection

Effect of spatial structure on epidemiology and evolution in the model.

<p>The top figures (A) show simulation results for the density of susceptible (dashed line) and infected hosts (full line) for different level of mixing (left: no mixing (<i>g</i>_<i>H</i> = <i>g</i>_<i>P</i> = 0), middle: intermediate (<i>g</i>_<i>H</i> = <i>g</i>_<i>P</i> = 0.5), right: full mixing (<i>g</i>_<i>H</i> = <i>g</i>_<i>P</i> = 1)). The figures in the middle (B) show simulation results for the change in mutant frequency in the total pathogen population (black), in the horizontally infected hosts (red) or in vertically infected hosts (blue) for different levels of mixing. The figures at the bottom (C) indicate the values of the different components of the selection coefficient identified in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004810#ppat.1004810.e003" target="_blank">Eq (1)</a>: the horizontal transmission term (red), the vertical transmission component (blue) and the sum of the previous two terms (black) and the magnitude of the cost of virulence (dashed black). Note that the global mutant frequency increases only when the sum of the transmission terms (full black line) is higher than the cost of virulence (dashed line). Our simulations are the result of the numerical integration of the deterministic approximation of the full spatial model, using Improved Pair Approximation (IPA; [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004810#ppat.1004810.ref031" target="_blank">31</a>]) with parameters <i>ϕ</i> = 1/6 and <i>θ</i> = 2/5, which correspond to a triangular lattice (each site has 6 neighbours, see [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004810#ppat.1004810.ref031" target="_blank">31</a>] for details). Parameters: <i>α</i>_<i>w</i> = 0.1, <i>α</i>_<i>m</i> = 1, <i>β</i>_<i>w</i> = 2, <i>β</i>_<i>m</i> = 3.5, <i>δ</i> = 0.99, <i>d</i> = 0.01. Initial conditions: <i>S</i>(0) = 0.8, </p><p></p><p></p><p></p><p><mi>I</mi><mi>w</mi><mi>H</mi></p><p><mo>(</mo><mn>0</mn><mo>)</mo></p><mo>=</mo><p><mi>I</mi><mi>m</mi><mi>H</mi></p><p><mo>(</mo><mn>0</mn><mo>)</mo></p><mo>=</mo><mn>0.01</mn><p></p><p></p><p></p>,<p></p><p></p><p></p><p><mi>I</mi><mi>w</mi><mi>V</mi></p><p><mo>(</mo><mn>0</mn><mo>)</mo></p><mo>=</mo><p><mi>I</mi><mi>m</mi><mi>V</mi></p><p><mo>(</mo><mn>0</mn><mo>)</mo></p><mo>=</mo><p></p><p><mn>10</mn></p><p><mo>−</mo><mn>5</mn></p><p></p><p></p><p></p><p></p>.<p></p

Competition of fluorescently marked virus (λCFP and λYFP—blue and green areas) during invasion into uninfected cells (black areas) at different degrees of mixing (<i>undisturbed</i>, <i>30s</i>, <i>24h</i>, <i>24h-wet</i>).

<p><b>(A)</b><i>Undisturbed environment</i>—Circular epidemic front segregates into single-virus sectors (blue and green sectors, white arrow depicts the centre of epidemic inoculation). <b>(B)</b><i>30s disturbance</i>—Epidemic clusters that are dominated by a single type of virus appear. <b>(C,D)</b> Longer periods of disturbance (<i>24h</i>) and a liquid surface layer (<i>24h-wet</i>) progressively homogenize the spatial genetic structure of virus types.</p

Mixing selects for virulence.

<p>A 1:1 mixture of temperate λ and its virulent mutant λcI857 was used to seed spatial epidemics. Spatial structure was disturbed to different degree (<b><i>undisturbed</i>, <i>30s</i>, <i>24h</i>, <i>24h-wet</i></b>) and transferred onto a fresh biofilm of susceptible hosts for 3 consecutive days. Disturbance of the epidemic structure has a strong effect on the competitive fitness of the virulent λcI857. The <b><i>undisturbed</i></b> environment strongly selects against virulence whereas in a well-mixed environment (<b><i>24h-wet</i></b>) virulence is beneficial. As expected, intermediate levels of mixing (<b><i>30s</i>, <i>24h</i></b>) lead to intermediate fitness of λcI857. Symbols represent 6 replications for each mixing treatment (with 3 independent replicates for each marker-mutant combination).</p

Effect of mixing on the relative contribution of horizontal (W_H) and vertical transmission (W_V) to the fitness of the virulent mutant in the model.

<p>Each curve corresponds to different levels of mixing: no mixing <i>g</i>_<i>H</i> = <i>g</i>_<i>P</i> = 0 (dotted line), intermediate mixing <i>g</i>_<i>H</i> = <i>g</i>_<i>P</i> = 0.5 (dashed line) and full mixing with <i>g</i>_<i>H</i> = <i>g</i>_<i>P</i> = 1 (full line). The contribution of horizontal transmission is maximal in the early stage of the epidemic because many susceptible hosts are still available. More mixing increases the contribution of horizontal transmission to the fitness of the mutant throughout the epidemics.</p

Schematic representation of the pathogen life cycle.

<p>In our model we assume that hosts can either be uninfected or infected by the wild type (in green) or a mutant genotype (in red). Transmission can take two different routes. Horizontal transmission occurs between a susceptible host and an infected host with genotype <i>i</i> with rate <i>β</i>_<i>i</i>[<i>I</i>|<i>S</i>] (see supporting information Theory in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004810#ppat.1004810.s001" target="_blank">S1 Text</a>). Vertical transmission occurs when and infected host reproduces in an empty site with rate <i>b</i>_<i>I</i><i>δq</i>_<i>0/i</i>, where <i>b</i>_<i>I</i> is the reproduction rate of infected hosts, <i>δ</i> is the fidelity of vertical transmission and <i>q</i>_<i>0/i</i> measures the average local density of empty sites around hosts infected with genotype <i>i</i>. Among hosts infected by the same genotype we distinguish hosts that got infected horizontally (noted </p><p></p><p></p><p></p><p><mi>I</mi><mi>i</mi><mi>H</mi></p><p></p><p></p><p></p>) or vertically (noted <p></p><p></p><p></p><p><mi>I</mi><mi>i</mi><mi>V</mi></p><p></p><p></p><p></p>). Both types of hosts can reproduce and have an increased mortality rate <i>α</i>_<i>i</i> (i.e. virulence) due to the infection with genotype <i>i</i>.<p></p

Effect of mixing on the relative contribution of horizontal (W_H) and vertical transmission (W_V) to the fitness of the virulent λcI857.

<p>More mixing (from <b><i>undisturbed</i>, <i>30s</i>, <i>24h</i></b> to <b><i>24h-wet</i></b>) increases the fitness contribution of horizontal transmission to the competitive ability of λcI857 (W_H) relative to the fitness contribution of vertical transmission (W_V) (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004810#ppat.1004810.g003" target="_blank">Fig 3</a>). Each box represents the median, the first and the third quartiles. Dashed lines delimit 1.5 times the inter-quartile range, above which individual counts are considered outliers and marked as empty circles.</p

Schematic representation of the bacteriophage λ life cycle.

<p>Free viral particles of the wild type virus (green) and the virulent mutant (red) infect susceptible cells . A proportion of successful infections leads to genome integration at rate and to produce infected cells and or results in cell lysis at rate and . Infected cells lyse through spontaneous reactivation of the provirus at rate and for and , respectively. (See Table S1 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003209#ppat.1003209.s009" target="_blank">Text S1</a> for the definition and the values of all the parameters of this model).</p