Dynamic ubiquitination drives herpesvirus neuroinvasion

Neuroinvasive herpesviruses display a remarkable propensity to enter the nervous system of healthy individuals in the absence of obvious trauma at the site of inoculation. We document a repurposing of cellular ubiquitin during infection to switch the virus between two invasive states. The states act sequentially to defeat consecutive host barriers of the peripheral nervous system and together promote the potent neuroinvasive phenotype. The first state directs virus access to nerve endings in peripheral tissue, whereas the second delivers virus particles within nerve fibers to the neural ganglia. Mutant viruses locked in either state remain competent to overcome the corresponding barrier but fail to invade the nervous system. The herpesvirus “ubiquitin switch” may explain the unusual ability of these viruses to routinely enter the nervous system and, as a consequence, their prevalence in human and veterinary hosts

The pUL37 tegument protein guides alphaherpesvirus retrograde axonal transport to promote neuroinvasion

A hallmark property of the neurotropic alpha-herpesvirinae is the dissemination of infection to sensory and autonomic ganglia of the peripheral nervous system following an initial exposure at mucosal surfaces. The peripheral ganglia serve as the latent virus reservoir and the source of recurrent infections such as cold sores (herpes simplex virus type I) and shingles (varicella zoster virus). However, the means by which these viruses routinely invade the nervous system is not fully understood. We report that an internal virion component, the pUL37 tegument protein, has a surface region that is an essential neuroinvasion effector. Mutation of this region rendered herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) incapable of spreading by retrograde axonal transport to peripheral ganglia both in culture and animals. By monitoring the axonal transport of individual viral particles by time-lapse fluorescence microscopy, the mutant viruses were determined to lack the characteristic sustained intracellular capsid motion along microtubules that normally traffics capsids to the neural soma. Consistent with the axonal transport deficit, the mutant viruses did not reach sites of latency in peripheral ganglia, and were avirulent. Despite this, viral propagation in peripheral tissues and in cultured epithelial cell lines remained robust. Selective elimination of retrograde delivery to the nervous system has long been sought after as a means to develop vaccines against these ubiquitous, and sometimes devastating viruses. In support of this potential, we find that HSV-1 and PRV mutated in the effector region of pUL37 evoked effective vaccination against subsequent nervous system challenges and encephalitic disease. These findings demonstrate that retrograde axonal transport of the herpesviruses occurs by a virus-directed mechanism that operates by coordinating opposing microtubule motors to favor sustained retrograde delivery of the virus to the peripheral ganglia. The ability to selectively eliminate the retrograde axonal transport mechanism from these viruses will be useful in trans-synaptic mapping studies of the mammalian nervous system, and affords a new vaccination paradigm for human and veterinary neurotropic herpesviruses

New tools to convert bacterial artificial chromosomes to a self-excising design and their application to a herpes simplex virus type 1 infectious clone

Background: Infectious clones are fundamental tools for the study of many viruses, allowing for efficient mutagenesis and reproducible production of genetically-defined strains. For the large dsDNA genomes of the herpesviridae, bacterial artificial chromosomes have become the cloning vector of choice due to their capacity to house full-length herpesvirus genomes as single contiguous inserts. Furthermore, while maintained as plasmids in Escherichia coli, the clones can be mutated using robust prokaryotic recombination systems. An important consideration in the design of these clones is the means by which the vector backbone is removed from the virus genome upon delivery into mammalian cells. A common approach to vector excision is to encode loxP sites flanking the vector sequences and rely on Cre recombinase expression from a transformed cell line. Here we examine the efficiency of vector removal using this method, and describe a “self-excising” infectious clone of HSV-1 strain F that offers enhancements in virus production and utility. Results: Insertion of a fluorescent protein expression cassette into the vector backbone of the HSV-1 strain F clone, pYEbac102, demonstrated that 2 serial passages on cells expressing Cre recombinase was required to achieve \u3e 95 % vector removal from the virus population, with 3 serial passages resulting in undetectable vector retention. This requirement was eliminated by replacing the reporter coding sequence with the CREin gene, which consists of a Cre coding sequence disrupted by a synthetic intron. This self-excising variant of the infectious clone produced virus that propagated with wild-type kinetics in culture and lacked vector attenuation in a mouse neurovirulence model. Conclusion: Conversion of a herpesvirus infectious clone into a self-excising variant enables rapid production of viruses lacking bacterial vector sequences, and removes the requirement to initially propagate viruses in cells that express Cre recombinase. The self-excising bacterial artificial chromosome described here allows for efficient production of the F strain of herpes simplex virus type 1

Proton pump inhibition also affects virion maturation.

<p>(A) H1-Hela cells were infected at an MOI of 50 pfu/cell, and half of the samples were treated with bafilomycin A1 as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003046#ppat-1003046-g004" target="_blank">Figure 4</a>. Cells were labeled with ³⁵S-Methionine from 3 h.p.i. until collection at 5 h.p.i., and lysates were then separated on a 15–30% sucrose gradients. Fractions were then collected and the counts per minute (CPM) were analyzed for each fraction. (B) Three fractions representing the 150S and 75S peaks were pooled and run on SDS-PAGE as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003046#ppat-1003046-g006" target="_blank">Figure 6</a>. The ratio of VP0 to VP3 bands was analyzed and plotted in arbitrary units. The titer of the infectious virus in the pooled 150S fractions was determined by plaque assay. * p<0.05.</p

Intracellular Vesicle Acidification Promotes Maturation of Infectious Poliovirus Particles

<div><p>The autophagic pathway acts as part of the immune response against a variety of pathogens. However, several pathogens subvert autophagic signaling to promote their own replication. In many cases it has been demonstrated that these pathogens inhibit or delay the degradative aspect of autophagy. Here, using poliovirus as a model virus, we report for the first time <em>bona fide</em> autophagic degradation occurring during infection with a virus whose replication is promoted by autophagy. We found that this degradation is not required to promote poliovirus replication. However, vesicular acidification, which in the case of autophagy precedes delivery of cargo to lysosomes, is required for normal levels of virus production. We show that blocking autophagosome formation inhibits viral RNA synthesis and subsequent steps in the virus cycle, while inhibiting vesicle acidification only inhibits the final maturation cleavage of virus particles. We suggest that particle assembly, genome encapsidation, and virion maturation may occur in a cellular compartment, and we propose the acidic mature autophagosome as a candidate vesicle. We discuss the implications of our findings in understanding the late stages of poliovirus replication, including the formation and maturation of virions and egress of infectious virus from cells.</p> </div

Intracellular poliovirus yields are not affected by lysosomal protease inhibitors.

<p>Triplicate samples of H1-Hela cells were infected with PV at an MOI of 0.1 pfu/cell (<b>A</b>) or MOI of 50 pfu/cell (<b>B</b>). Cells were pre-treated with 20 µM leupeptin for 14 hours prior to infection and kept under treatment throughout infection. Cell-associated virus was collected at the indicated times post-infection, and virus titers were determined by plaque assay. Parallel infections were collected for Western blots of LC3 (A) or p62 (B) In (B) cell-associated virus was collected at 6 h.p.i. for plaque assay. (<b>C</b>) Infections were performed as in (A), then 10 µg/mL each of E64d and Pepstatin A (Pep.A) were added to the media at the time of infection. Inhibition of lysosomal degradation was confirmed by immunoblot for LC3 and GAPDH. (<b>D</b>) Infection at an MOI of 50 pfu/cell, with E64/Pep.A as in (C).</p

Degradation of p62 is dependent on the induction of autophagy.

<p>(A) Cells were transfected with scrambled (SCR) or anti-LC3 (LC3) siRNAs, and 48 h later infected with PV at an MOI of 50 pfu/cell, or mock infected. Cells were collected at 6 h.p.i., and immunoblots were performed on lysates for p62, GAPDH, and LC3. (B) Cells were treated with 10 mM 3-MA for 12 h, then infected with PV at an MOI of 50 pfu/cell, or mock infected. Cells were collected at 6 h.p.i. and lysates were used for immunoblots.</p

Intracellular poliovirus yields are reduced when cells are treated with inhibitors of vesicle acidification.

<p>(A) Triplicate samples of H1-Hela cells were infected with PV at an MOI of 0.1 pfu/cell, and cell-associated virus was collected at the indicated times post-infection. NH₄Cl (20 mM) was added to the media at the time of infection (solid line). Virus titers were then determined by plaque assay. (B) Infection as in (A), carried out to 16 h to represent a multiple-cycle infection. (C) Triplicate H1-Hela infections with PV at an MOI of 50 pfu/cell, with cell-associated virus collected at 6 h.p.i. (D) Cells were infected as in (A), then NH₄Cl was added to half of the samples at 3.5 hours post-infection, and cell-associated virus was collected immediately or at 7 h.p.i. (E) Cells were pre-treated for 14 hours with 0.1 µM bafilomycin A1 (Baf.A1) and kept under treatment throughout infection (MOI = 0.1 pfu/cell). Cell-associated virus was collected at 6 or 12 h.p.i. and virus titers were determined by plaque assay. * p<0.05, ** p<0.01, *** p<0.0001.</p

Poliovirus entry, translation, and RNA replication are unaffected by treatment with inhibitors of vesicle acidification.

<p>(<b>A</b>) H1-HeLa cells were infected with PV at an MOI of 50 pfu/cell. Cells were pulsed at the indicated h.p.i with ³⁵S-labeled methionine for 1 h then lysed. Lysates were run on SDS-PAGE. Expected viral proteins are labeled according to recognized banding patterns. (<b>B</b>) Triplicate plates of H1-Hela cells were infected with PV at an MOI of 0.1 pfu/cell, virus RNA and host GAPDH RNA were measured by qRT-PCR. Virus RNA levels were normalized to GAPDH levels using the delta-Ct method. NH₄Cl treatment was as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003046#ppat-1003046-g004" target="_blank">Figure 4</a>, and Guanidine HCl (2 mM) was added to the media at the time of infection. The data shown are pooled from three replicate experiments, and the titer of cell-associated virus collected at 6h.p.i. from each replicate was determined by plaque assay. (<b>C</b>) Triplicate plates of 293T cells were treated with 20 mM 3-MA for 2 hours prior to infection, and kept under treatment throughout infection. PV infections were done at an MOI of 0.1 pfu/cell. RNA levels and virus titers were analyzed as in (B). ** p<0.01, *** p<0.0001.</p

Virion maturation requires acidic compartments.

<p>(<b>A</b>) H1-Hela cells were infected at an MOI of 50 pfu/cell, and half of the samples were treated with NH₄Cl. Cells were labeled with ³⁵S-Methionine from 3 h.p.i. until collection at 5 or 6 h.p.i., and lysates were then separated on a 15–30% sucrose gradients. Fractions were then collected and the counts per minute (CPM) were analyzed for each fraction. Representative gradients from three independent experiments are shown. (<b>B</b>) The three fractions representing the 150S peak in each experiment were pooled for plaque assay analysis. Data shown are the averages from three independent experiments. (<b>C</b>) Three fractions representing the 150S and 75S peaks were pooled and run on SDS-PAGE, and the ³⁵S-Methionine labeled bands were visualized. The bands are labeled according to expected relative migration pattern, and VP2 is identified by its absence in the 75S peak. The ratio of VP0 to VP3 bands was analyzed from four independent experiments and plotted in arbitrary units. * p<0.05, ** p<0.01, *** p<0.0001.</p