(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



Dysregulated metabolism of the late herpes simplex virus 1 transcriptome through the vhs-VP22 axis uncouples virus cytopathic effect and virus production [1]

['Kathleen Pheasant', 'Section Of Virology', 'Department Of Microbial Sciences', 'University Of Surrey', 'Guildford', 'United Kingdom', 'Dana Perry', 'Emma L. Wise', 'Vivian Cheng', 'Gillian Elliott']

Date: 2023-06

Herpes simplex virus 1 (HSV1) expresses its genes in a classical cascade culminating in the production of large amounts of structural proteins to facilitate virus assembly. HSV1 lacking the virus protein VP22 (Δ22) exhibits late translational shutoff, a phenotype that has been attributed to the unrestrained activity of the virion host shutoff (vhs) protein, a virus-encoded endoribonuclease which induces mRNA degradation during infection. We have previously shown that vhs is also involved in regulating the nuclear-cytoplasmic compartmentalisation of the virus transcriptome, and in the absence of VP22 a number of virus transcripts are sequestered in the nucleus late in infection. Here we show that despite expressing minimal amounts of structural proteins and failing to plaque on human fibroblasts, the strain 17 Δ22 virus replicates and spreads as efficiently as Wt virus, but without causing cytopathic effect (CPE). Nonetheless, CPE-causing virus spontaneously appeared on Δ22-infected human fibroblasts, and four viruses isolated in this way had all acquired point mutations in vhs which rescued late protein translation. However, unlike a virus deleted for vhs, these viruses still induced the degradation of both cellular and viral mRNA suggesting that vhs mutation in the absence of VP22 is necessary to overcome a more complex disturbance in mRNA metabolism than mRNA degradation alone. The ultimate outcome of secondary mutations in vhs is therefore the rescue of virus-induced CPE caused by late protein synthesis, and while there is a clear selective pressure on HSV1 to mutate vhs for optimal production of late structural proteins, the purpose of this is over and above that of virus production.

HSV is a human pathogen that lytically infects cells of the epidermis. Following viral genome replication, structural proteins are produced in abundance to enable the rapid assembly and release of large quantities of infectious progeny. Infected cells also exhibit cytopathic effect (CPE), morphological changes that are exemplified by cell rounding and the breakage of cell-to-cell contacts, facilitating virus dissemination. Here we show that HSV1 with a mutation that results in shutdown of late protein synthesis also fails to cause CPE. However, unexpectedly, we found that this virus is still able to release large numbers of infectious virus which can spread between cells without any evidence of cell damage. Nonetheless, despite efficient virus productivity, this virus spontaneously mutates to rescue late protein production and CPE, with mutations that result in altered RNA metabolism. There is therefore a clear selective pressure on HSV1 to optimize the synthesis of late structural proteins, but the purpose of this is over and above that of virus production, a result that has implications for why viruses in general express such large amounts of structural proteins.

Here we report the unexpected result that despite translational shutoff and lack of plaque formation in primary human fibroblasts, HSV1 lacking VP22 replicates, spreads and produces as much infectious progeny virus as Wt virus without causing any cytopathic effect (CPE) in these cells. Nonetheless, CPE-inducing virus rapidly appeared as plaques in Δ22-infected fibroblasts and these rescued viruses had all acquired mutations in vhs which restored late protein translation. This suggests that despite efficient virus propagation in the absence of VP22, there is pressure on the virus to mutate vhs to rescue late protein translation and concomitant CPE, over and above what is required for virus production. These results have implications for understanding why this and potentially other viruses express such large amounts of late virus proteins.

A clue to the mechanism of late translational shutoff came from the observation that spontaneous secondary mutations frequently arise in the UL41 gene of the Δ22 genome [ 4 , 6 , 7 ], a gene which encodes the virion host shutoff (vhs) protein [ 8 ]. These mutations rescue the deleterious effect of VP22 deletion on late protein translation, restoring plaque formation [ 4 , 6 , 7 ]. The vhs protein is an endoribonuclease which induces the degradation of cellular mRNA during HSV1 infection through its endoribonuclease cleavage of cytoplasmic mRNAs followed by Xrn1 exonuclease degradation [ 9 ], and regulates the transition from IE to E and L gene expression [ 10 , 11 ]. It was therefore originally proposed that VP22 is required to quench vhs-induced mRNA degradation at later times in infection and that in the absence of VP22, vhs endoribonuclease activity is lethal [ 6 ]. Nonetheless in our hands, infection of human fibroblasts with a Δ22 virus did not result in unrestrained mRNA degradation compared to Wt infection [ 4 ]. Moreover, in that study we also demonstrated that in Wt infection, IE and E transcripts were concentrated in the nucleus at late times, but in cells infected with a Δvhs virus all classes of transcripts were present in the cytoplasm [ 4 ], suggesting that the vhs endoribonuclease may be involved in regulating mRNA localisation, and providing a link between mRNA degradation in the cytoplasm and mRNA retention in the nucleus. The relative compartmentalisation of the virus transcriptome was also mirrored by the localisation of the polyA binding protein PABPC1, a protein that has a steady-state cytoplasmic localisation but shuttles between the cytoplasm and nucleus to bind polyadenylated mRNAs [ 12 , 13 ]. Once mRNA in the cytoplasm has been turned over, PABPC1 recycles back to the nucleus, and in the presence of functional vhs, PABPC1 accumulates there in an endoribonuclease dependent fashion [ 14 , 15 ].

Herpes simplex virus type 1(HSV1) expresses its genes in a classical cascade of gene expression during lytic infection, comprising immediate-early, early and late genes [ 1 ]. In general, the late genes encode virus structural proteins and are transcribed predominantly from replicated DNA genomes, leading to a large burst of late protein synthesis for optimal virus assembly. Deletion of the HSV1 UL49 gene which encodes the tegument protein VP22 [ 2 ] results in a virus that exhibits translational shutoff of late protein synthesis [ 3 , 4 ], and in many systems is detrimental to virus propagation [ 4 – 6 ]. This translational shutoff is not a consequence of enhanced host responses such as the stress response kinase protein kinase R. Rather, it correlates with an increased nuclear accumulation of viral transcripts in cells infected with the Δ22 virus, as demonstrated in our previous studies using mRNA FISH, thereby preventing their translation in the cytoplasm [ 4 ].

Results

HSV1 lacking VP22 replicates and spreads in primary human fibroblast cells without causing cytopathic effect Deletion of the VP22-encoding gene (UL49) has been shown to be detrimental to HSV1, resulting in late translational shutoff [4,6,7]. Our own Δ22 virus based on strain 17 fails to plaque on primary human fibroblasts (HFFF) as late as 5 dpi (Fig 1A). The efficiency of viral DNA replication in Δ22 infection was measured by harvesting at 2 or 16 h after infection and determining the relative level of viral DNA by qPCR of the virus gene UL48, to reflect input viral DNA (2 h) or viral DNA replication (16 h). Although there was less input DNA in Δ22 infected cells, the relative increase in genome copies was similar in Wt and Δ22 infected cells at 16 h, indicating that the absence of VP22 has little effect on genome replication (Fig 1B), and that the block to virus production occurs at a later stage. Western blotting of HFFF cells infected at high multiplicity confirmed that a range of virus envelope proteins are poorly expressed (Fig 1C), in line with the previously demonstrated translational shutoff in these cells [4], providing an obvious explanation for the inability of this virus to form plaques in HFFF cells. Immunofluorescence of infected cells revealed that the IE protein ICP4, which localises in a distinctive cytoplasmic punctate pattern late in Wt infection, was restricted to the nucleus in Δ22 infected cells (Fig 1D, ICP4) while the envelope protein glycoprotein E (gE) was concentrated in a juxtanuclear compartment rather than progressing to the plasma membrane as it does in Wt infection (Fig 1D, gE). These results suggest there is a block to late protein trafficking in the absence of VP22. However, despite these obvious defects in Δ22 infection, we found no significant difference in the total virus produced by Δ22 compared to Wt virus in HFFF in a one-step growth curve (Fig 1E). Moreover, low magnification imaging of HFFF cells infected with Δ22 (which expresses GFP in place of VP22) showed that while all cells were GFP positive after 20 h, there was no sign of the classical HSV1-induced cytopathic effect (CPE) of cell-rounding, which was evident in cells infected with Wt or HSV1 expressing GFP fused to VP22 (GFP-22) infected cells (Fig 1F). By contrast, HSV1 expressing GFP in place of UL34, a protein essential for nuclear egress [16], exhibited CPE similar to Wt and GFP-22 (Fig 1F), indicating that even though this virus is unable to export capsids to the cytoplasm or assemble progeny virions, it is still able to cause CPE. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. HSV1 replicates in primary human fibroblasts in the absence of VP22 without causing CPE. (A) HFFF cells were infected with approximately 50 pfu of Wt s17 and Δ22 viruses, fixed at 2, 3, 4 and 5 days (dpi) and stained with crystal violet. (B) HFFF cells were infected with Wt s17 or Δ22 virus at a multiplicity of 3, acid washed at 1 hpi, then DNA was isolated at 2 or 16 hpi. qPCR was performed for gene UL48 to determine the relative virus DNA copy number represented as ΔΔCt to s17 infection in the presence of AraC (mean±SEM, n = 3). (C) HFFF cells infected with Wt (s17) or Δ22 viruses at MOI 2 were harvested at 16 hpi and analysed by SDS-PAGE and Western blotting with antibodies as indicated. (D) HFFF cells infected with Wt (s17) or Δ22 viruses at MOI 2 were fixed at 16 hpi and analysed by immunofluorescence with antibodies to the IE protein ICP4 and the L protein glycoprotein E (gE), both in green. Nuclei were stained with DAPI (blue). Scale bar = 50 μm. (E) HFFF cells were infected with Wt s17 or Δ22 virus at a multiplicity of 2, total virus harvested every 5 h up to 20 h and titrated onto Vero cells (mean±SEM, n = 3). (F) HFFF cells grown in 6-well plates were left uninfected (mock) or infected at a multiplicity of 2 with Wt s17, HSV1 GFP-22, Δ22 expressing GFP or Δ34 expressing GFP. After 20 h the cells were imaged live using brightfield and fluorecence where appropriate. Scale bar = 100 μm. https://doi.org/10.1371/journal.ppat.1010966.g001 Given that the Δ22 virus does not plaque on HFFF, we next investigated its ability to spread in these cells. A multi-step growth curve was carried out by infecting HFFF cells at a multiplicity of 0.01 and intriguingly, this also revealed little difference in the replication or release of Wt and Δ22 viruses, in a scenario where optimal virus replication requires multiple rounds of replication and spread to other cells in the monolayer (Fig 2A). GFP imaging of cells infected at low multiplicity revealed that the entire monolayer of cells had become GFP positive but without causing CPE, indicating that the Δ22 virus spreads efficiently without affecting the integrity of the cells (Fig 2B). To further visualise the behaviour of the Δ22 virus at low multiplicity and determine if the virus can spread cell-to-cell, HFFF cells were infected with Δ22 (which expresses GFP in place of VP22) or HSV1 GFP-22 at around 20 pfu per well in the presence of 1% human serum to block extracellular virus [17]. Brightfield and GFP fluorescence of representative fields were imaged up to 3 days after infection to investigate virus spread. While HSV1 expressing GFP-22 was seen to spread over time, causing the rounding up of cells as expected (Fig 2C, GFP22), Δ22 failed to cause any obvious CPE (Fig 2C, Δ22, brightfield). Nonetheless, GFP fluorescence was detectable in a cluster of cells at day one, spreading into a much larger area over the next two days (Fig 2C, Δ22, GFP). By contrast, HFFF cells infected with the Δ34 virus at low multiplicity exhibited only individual rounded up cells as late as 3 days after infection, as would be expected as this virus is unable to produce progeny virions (Fig 2D, Δ34). Taken together, these results suggest that despite extreme translational shutoff and no obvious virus-induced pathology, the absence of VP22 has little effect on the propagation of HSV1 in HFFF cells. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. HSV1 spreads in the absence of VP22 without causing CPE. (A) HFFF cells were infected with Wt s17 or Δ22 virus at a multiplicity of 0.01, supernatant (s/n) and cell-associated (c/a) virus harvested every day for 4 days and titrated onto Vero cells (mean±SEM, n = 3). (B) HFFF cells were infected with approximately 20 pfu of HSV1 GFP- Δ22 virus and brightfield and GFP images acquired 3 days later. Scale bar = 100 μm. (C) HFFF cells were infected with approximately 20 pfu of HSV1 GFP-22 or Δ22 viruses in the presence of 1% human serum and representative brightfield and GFP images acquired every day for 3 days. Scale bar = 100 μm. (D) Confluent HFFF cells were infected with approximately 20 pfu of Δ34 virus and representative brightfield and GFP images acquired at days 1 and 3. Scale bar = 100 μm. https://doi.org/10.1371/journal.ppat.1010966.g002

Point mutations in vhs rescue translational shutoff in Δ22 infected cells Although the Δ22 virus does not plaque on HFFF cells, plaques spontaneously appear on HFFF cells at a rate of ~ 1 in every 100 pfu, as judged by the original titre on Vero cells [4]. Further analysis of one of these viruses (Δ22*) had previously revealed a single point mutation in the vhs open reading frame (A95T) which had rescued both translation and plaque formation [4]. Taken together with studies from other groups, which have described the rescue of Δ22 replication through spontaneous mutation of vhs [6,7], and single residue changes in vhs having a profound effect on its activity [18,19], these results led us to initially hypothesize that the A95T mutation had inactivated the vhs endoribonuclease activity, thereby rescuing late protein synthesis and subsequent virus replication. We have now undertaken a more extensive analysis of this and three additional rescued viruses that were isolated from plaques on HFFF and which formed plaques approaching the size of Wt plaques (Fig 3A). They all express full-length vhs as demonstrated by Western blotting (Fig 3B), indicating that no gross mutations had occurred within the vhs open reading frame, but metabolic labelling profiles confirmed that all these viruses had rescued the translational shutoff exhibited by the Δ22 virus, albeit the PP13 virus recovering only slightly from the Δ22 base line (Fig 3C). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. Point mutations in the vhs endoribonuclease rescue plaque formation and translation of Δ22 viruses. (A) Virus from four plaques that appeared spontaneously on Δ22-infected HFFF cells was purified and plated onto HFFF cells at around 50 pfu per well (as judged by titre on Vero cells). After 3 days, cells were fixed and stained with crystal violet. (B) HFFF cells were infected with the indicated viruses at a multiplicity of 2, harvested at 16 h, subjected to SDS-PAGE and Western blotting for VP22, GFP, vhs and α-tubulin and images acquired with a LICOR Odyssey imaging system. (C) HFFF cells were infected with the indicated viruses at a multiplicity of 2, and 16 hours later were incubated in the presence of [35S]-methionine for a further 60 mins. The cells were then lysed and analysed by SDS-PAGE followed by autoradiography. (D) A line drawing of the vhs open reading frame indicating the point mutations found in the vhs gene of the rescued Δ22 viruses. The vhs-encoding gene, UL41, was amplified by PCR from the submaster stock of our Δ22 virus using four amplicons to cover the entire gene. These amplicons were sequenced by NGS (~40,000 sequences per amplicon) and all variations to the published strain 17 reference sequence (NC001806) scored as the percentage present in the population. https://doi.org/10.1371/journal.ppat.1010966.g003 Sequencing of the UL41 gene in these rescue viruses revealed that they all had point-mutations in the vhs open reading frame (Fig 3D). To determine if these variants were present in our original Δ22 virus stock or had arisen during propagation on HFFF cells, we carried out next generation sequencing of four amplicons covering the UL41 gene generated from the genome of our Δ22 virus stock (Fig 3D), revealing that it already contained the T102M and V271A variations at a rate of 33% and 35% respectively, with I223V at a much lower rate of 2% (Fig 3D). No V33A or A95T variations were found by deep sequencing this virus suggesting that they may have arisen spontaneously during propagation on HFFF. Interestingly, direct sequencing of the UL41 gene from 16 viruses isolated from plaques on Vero cells in which this virus is able to plaque, or from nine non-CPE fluorescent foci on HFFF such as those shown in Fig 2C, showed that all viruses contained either the T102M or the V271A variation but none of them contained two mutations. This suggests that each of these single variations in isolation was not sufficient to rescue CPE of this virus in HFFF, and that, with the exception of the A95T variation, a second point mutation was required. To further ensure that no additional secondary mutation had occurred elsewhere in these four rescue viruses that could explain their phenotype, we carried out next generation sequencing of the Δ22*, PP12, PP13 and PP15 genomes and compared them to our previously published sequence for the Δ22 virus [20]. On average each of the viruses had gained five coding changes generally in the form of single amino acid changes, none of which provide an obvious explanation for the behaviour of these viruses (Table 1). Nonetheless, it is noteworthy that the Δ22* genome contained apparent insertions in UL26 and UL27, which although unlikely to explain a difference in relative RNA metabolism, are worth exploring. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Table 1. Coding changes found in the genome sequences of the four Δ22 rescue viruses. Genome sequences were aligned with the strain 17 reference sequence (JN555585.1). Coding changes from the parental Δ22 virus are highlighted in bold. https://doi.org/10.1371/journal.ppat.1010966.t001

Relative compartmentalisation of viral transcripts in cells infected with Δ22 rescue viruses Given that our previous study had indicated differential compartmentalisation of the virus transcriptome in Δ22 infected cells, we next investigated the subcellular localisation of E (TK) and L (gD) transcripts in HFFF cells infected with the Δ22 rescue viruses by mRNA FISH at 16 hours after infection. As shown previously [4], the IE transcript TK but not the L transcript gD was retained in the nucleus of Wt infected cells, while both transcripts were cytoplasmic in the absence of vhs (Fig 4A). By contrast and as shown before [4], both transcripts were almost entirely nuclear in Δ22 infected cells, thereby explaining the observed translational shutoff seen in these cells. In the case of the rescue viruses, virus transcript localisation ranged from both being completely cytoplasmic (Fig 4A, Δ22*) similar to that seen in Δvhs infection, to compartmentalisation patterns that were minimally altered compared to Δ22 (Fig 4A, PP13 and PP15). Quantification of the nuclear gD and TK transcript levels in a second FISH experiment confirmed that there was up to 3-fold more of the IE TK transcript in Wt infected nuclei compared to Δvhs infected nuclei, and up to 5-fold more in nuclei of Δ22 infected cells (Fig 4B). In the case of the late gD transcript, the level was similar between Wt and Δvhs infected nuclei but 2-fold more for Δ22 infected cells, while of the four Δ22 rescue viruses, only the Δ22* virus exhibited nuclear transcript levels as low as those found in Δvhs infected cells. Nonetheless, in all cases there were detectable levels of gD transcripts in the cytoplasm of the rescued virus-infected cells suggesting that vhs mutation in the Δ22 virus allowed the cytoplasmic localisation of sufficient late transcripts to increase late protein translation. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. Localisation of viral transcripts in cells infected with Δ22 rescue viruses. (A) HFFF cells grown in two-well slide chambers were infected with the viruses as indicated at MOI 2 and fixed after 16 hours in 4% paraformaldehyde. Cells were then processed for mRNA FISH using probes specific for E (TK in red) and L (gD in green) transcripts. Nuclei were stained with DAPI (blue). Scale bar = 20μm. (B) Cells treated in the same manner as (A) were imaged by confocal microscopy using a narrow pinhole, and the mean intensity of gD an TK transcript signal was measured using NIH ImageJ. The representative results of five nuclei are shown. Statistical analysis was carried out using an ordinary one-way ANOVA and comparison made to Dvhs values. ns, p > 0.05. * p < 0.05. ** p < 0.01. *** p < 0.001. https://doi.org/10.1371/journal.ppat.1010966.g004 In uninfected cells, PABPC1 has a steady state cytoplasmic localisation, but shuttles between the nucleus and the cytoplasm, binding the polyA tail of mRNAs in the nucleus and being transported out on those tails. It then returns to the nucleus after mRNA turnover in the cytoplasm to be exported again [12]. We have previously shown that in the absence of VP22, the cellular polyA binding protein PABPC1 accumulates to high levels in the nucleus [4,14], a result we had postulated to be the consequence of the aforementioned nuclear retention of late viral mRNA. We therefore examined the relative compartmentalisation of PABPC1 in HFFF cells infected with Wt, Δvhs, Δ22 or rescue viruses at 10 and 16 hours after infection. As shown before, PABPC1 had partially accumulated in the nucleus of Wt infected cells at 16 h, but remained cytoplasmic in Δvhs infected cells throughout, confirming the role that vhs plays in nuclear relocalisation of PAPBC1 (Fig 5). By contrast, in Δ22 infected cells, PABPC1 had already accumulated in nuclei by 10 h, and was almost entirely nuclear by 16 h, correlating with the extensive accumulation of viral mRNA seen at this time (Fig 5, Δ22). As we have shown before that vhs-induced degradation of mRNA is delayed rather than unrestrained in Δ22 infected HFFF cells [4], this early accumulation of PABPC1 in the nucleus is not a consequence of enhanced degradation of mRNA leading to more PABPC1 entering the nucleus. The relative nuclear accumulation of PABPC1 in cells infected with the Δ22 rescue viruses closely reflected the mRNA nuclear retention seen above: the Δ22* infection was similar to Δvhs, with no nuclear PABPC1; the PP15 virus was similar to Wt, with some PABPC1 in the nucleus; and the two other viruses (PP12 and PP13) caused nuclear accumulation of PABPC1 at levels somewhere between Wt and Δ22 (Fig 5). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 5. Relative compartmentalisation of PABPC1 in HFFF cells infected with Δ22 rescue viruses. HFFF cells infected with the indicated viruses at MOI 2 were fixed at 10 h and 16 h, stained with an antibody for PABPC1 (green) and nuclei stained with DAPI (blue). Scale bar = 50 μm. https://doi.org/10.1371/journal.ppat.1010966.g005

[END]
---
[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010966

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.