The herpes simplex virus 1 Us3 kinase is involved in assembly of membranes needed for viral envelopment and in distribution of glycoprotein K

Background : Capsids of herpes simplex virus 1 (HSV-1) are assembled in cell nuclei, released into the perinuclear space by budding at the inner nuclear membrane acquiring tegument and envelope. Alternatively, capsids gain access to the cytoplasm via dilated nuclear pores. They are enveloped by Golgi membranes. Us3 is a non-essential viral kinase that is involved in nucleus-to-cytoplasm translocation, preventing apoptosis and regulation of phospholipid-biosynthesis. Us3-deletion mutants (HSV-1∆Us3) accumulate in the perinuclear space. Nuclear and Golgi membranes proliferate, and homogeneous, proteinaceous structures of unknown identity are deposited in nuclei and cytoplasm. Glycoprotein K (gK), a highly hydrophobic viral protein, is essential for production of infectious progeny virus but, according to the literature, exclusively vital for envelopment of capsids by Golgi membranes. In the absence of Us3, virions remain stuck in the perinuclear space but mature to infectivity without reaching Golgi membranes, suggesting further function of gK than assumed. Methods : We constructed a HSV-1∆Us3 mutant designated CK177∆Us3gK-HA, in which gK was hemagglutinin (HA) epitope-tagged in order to localize gK by immunolabeling using antibodies against HA for light and electron microscopy. Results : CK177∆Us3gK-HA-infected Vero cells showed similar alterations as those reported for other HSV-1∆Us3, including accumulation of virions in the perinuclear space, overproduction of nuclear and Golgi membranes containing electron dense material with staining property of proteins. Immunolabeling using antibodies against HA revealed that gK is overproduced and localized at nuclear membranes, perinuclear virions stuck in the perinuclear space, Golgi membranes and on protein deposits in cytoplasm and nuclei. Conclusions : Us3 is involved in proper assembly of membranes needed for envelopment and incorporation of gK. Without Us3, virions derived by budding at nuclear membranes remain stuck in the perinuclear space but incorporate gK into their envelope to gain infectivity.

Generation of recombinants CK177ΔUs3gK-HA and CK177gK-HA Recombineering of pYEbac102 (Tanaka et al., 2003) was done in two steps in Escherichia coli strain SW102 (Warming et al., 2005) First, a galK expression cassette was amplified by PCR (Phusion R, High-Fidelity DNA Polymerase; M0530L, BioLabs. Ipswich, MA, USA, according to manufacturer's recommendations) with homology arms flanking UL53 using the following primers: primer forward, 5'-tct tcg gtg cca gtc cgc tgc acc gat gta ttt acg cgg tac gcc cca ccg CCT GTT GAC AAT TAA TCA TCG GCA -3'; primer reverse, 5'-gtt tcc aat ttg cat atg ccg tta cgg ttt ccg ccg gcc tgg atg tga cgt TCA GCA CTG TCC TGC TCC TT -3' (binding sequences in capitals). This amplimer was DpnI treated to remove template DNA, purified, and electroporated into competent and induced E. coli strain SW102 carrying pYEbac102 (Tanaka et al., 2003). Recombinant colonies were selected for growth on galactose plates. The BAC DNA carrying the HSV genomic sequence with deleted UL53 was designated pYEbac102ΔUL53. Second, a kanamycin resistant cassette was amplified by PCR on pBSrspL (Genes Bridges, Dresden, Germany; kanamycin resistant cassette) with the following primers for Us3rpsL: primer forward, 5'-ctt ccc aca cca cac cac cca gcg agg ccg agc gcc tgt gtc atc tgc aGG GCC TGG TGA TGA TGG CGG GAT CG-3', primer reverse 5'-aga tca cca gac cgg cgc tcc aaa tgt cga cgg tcg tgg tat acg gat ccT CAG AAG AAC TCG TCA AGA AGG-3' (binding sequences in capitals). The primers were chosen to yield the same deletion of Us3 as described by Purves et al. (1987). This amplimer was DpnI-treated, purified, and electroporated into competent and induced E. coli strain SW102 carrying pYEbac102ΔUL53. Recombinant colonies were selected for growth on kanamycin plates. Finally, fHSVgKgalKΔUS3, pCS177 (HA tagged gK and flanking sequences, see below) and pCMV-Cre (Cre recombinase under CMV promoter) DNA was mixed and co-transfected into Vero cells using Lipofectamine 2000 (11668027, Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer's recommendations. Rescued progeny virus with deleted Us3 and BAC backbone but HA-tagged gK, designated CK177ΔUs3gK-HA, was propagated in Vero cells.
Plasmid pCS177 containing the UL53 gene with a carboxy terminal HA tag and flanking sequence to UL53 that extend 0.3 kbp upstream and 0.4 kbp downstream of the UL53 gene was constructed as follows. First, downstream sequence of UL53 was amplified by PCR from wtHSV-1 genome (primer forward, 5'-gat ctc tag acg tca cat cca ggc cgg cgg aa -3'; primer reverse, 5'-gat cga gct cag gCC TCC GGC ACA GAC AAG GAC CAA T -3'; HSV-1 sequences in capitals). The resulting PCR product was digested with SacI and XbaI and cloned into the SacI and XbaI sites in pBluescript II KS(+), resulting in plasmid pCS176. Second, the UL53 gene with its upstream flanking sequence was amplified by PCR from wtHSV-1 using a reverse primer containing nucleotides of HA tag (primer forward, 5'-gat caa gct tag gcc tgg gtc ggt aca acg tac agc cgg at -3'; primer reverse, 5'-gat ctc tag aTC Acc atg gag cat aat ctg gaa cat cat atg gat aTA CAT CAA ACA GGC GCC TCT gga -3'; HSV-1 sequences in capitals). Following PCR, the DNA product was digested with HindIII and XbaI and inserted into these sites in pCS176, resulting in plasmid pCS177. The StuI fragment of pCS177 containing UL53-HA gene with flanking sequence was co-transfected together with pYEbac102ΔUL53 and pCMV-Cre into Vero cells. gK-HA expression was identified using indirect immunofluorescence and one virus stock expressing gK-HA was designated CK177gK-HA.

Infection of cells
Vero cells were grown for 2 days on cover slips (Assistent, Sondheim, Germany) for immunofluorescence, on sapphire disks (100.00174, Bruegger, Minusion, Switzerland) placed in 6 well plates for TEM, or in 75 cm 2 cell culture flasks for immunogoldlabeling, for 2 days prior to inoculation with CK177ΔUs3gK-HA, R7041ΔUs3, CK177gK-HA or wtHSV-1 at a multiplicity of infection (MOI) of 1 plaque-forming unit (pfu)/ml.

Cryo-fixation for transmission electron microscopy
Cells on sapphire disks were frozen 16 to 24 hpi in a high-pressure freezer (HPM010; BAL-TEC Inc., Balzers, Liechtenstein) and prepared for sectioning as described in detail previously (Wild et al., 2018;Wild et al., 2002). Cells were analyzed in a transmission electron microscope (CM12; FEI, Eindhoven, The Netherlands) equipped with a CCD camera (Ultrascan 1000; Gatan, Pleasanton, CA, USA).

Immunogold labeling
Inoculated cells were harvested at 20 hpi and prepared according to (Tokuyasu, 1973;Tokuyasu, 1980). Cells fixed with 4% formaldehyde in 0.1 M Na-phosphate, pH 7.4, for 2 h at room temperature were scraped from the flasks, washed three times with 0.1 M Na-phosphate by centrifugation at 13,000g for 30 s, and pelleted in 12% gelatine by centrifugation at 13,000g for 3 min at 37°C. After gelation at 4°C, 1 mm 3 blocks were immersed overnight in 2.3 M sucrose constantly rotating at 4°C. The infiltrated blocks were mounted on specimen holders, frozen by plunging into liquid nitrogen, and placed into the cryo-microtome (UC6, Leica, Vienna, Austria) at -120°C. Ultra-thin sections of 80-100 nm were collected on carbon-coated formvar films mounted on hexagonal 100 mesh/inch copper grids. Sections were washed by floating on several drops of buffer and blocking solutions prior to routine labeling procedure (Slot et al., 1991) with primary antibodies (1:30) against HA (SC-805, Santa Cruz Biotechnology), and secondary anti-rabbit antibodies (1:30) coupled to 12 nm colloidal gold (111-205-144, Jackson ImmunoResearch, West Grove, PA, USA). After labeling, sections were washed on distilled water droplets, stained by immersing in a mixture of 1.8% methyl cellulose and 0.4% uranyl acetate (Griffiths et al., 1983) for 10 min, and dried for imaging by TEM (Philips, CM12). For controls, primary antibodies were omitted, and labeling was also performed in wtHSV-1 infected cells.

Results
CK177ΔUs3gK-HA induces alteration of nuclear and Golgi membranes Infection with HSV-1ΔUs3 induces formation of folds and invagination of nuclear membranes associated with accumulation of virions (Poon et al., 2006;Reynolds et al., 2002;Wisner et al., 2009). Infection with CK177ΔUs3gK-HA, a similar mutant lacking Us3 but equipped with an epitope-tagged gK, also induced multiple folds and invaginations of nuclear membranes containing virions. Moreover, homogenous, proteinaceous structures occurred within nuclei and cytoplasm of CK177ΔUs3gK-HA ( Figure 1) and R7041ΔUs3 (Figure 2A  gK localizes on Golgi membranes, nuclear membranes, virions, and proteinaceous deposits Budding of capsids at nuclear and Golgi membranes starts by insertion of budding proteins that appears in electron micrographs as a dense layer (Leuzinger et al., 2005). The major budding proteins at nuclear membranes are UL31/UL34 (Bigalke et al., 2014;Hagen et al., 2015), while gK and UL20 protein are responsible for budding at Golgi membranes (Melancon et al., 2007). Infection with CK177gK-HA revealed that gK localizes in cytoplasm, nuclear rim and even in nuclei ( Figure 4A) by immunofluorescence using antibodies against HA. Following infection with CK177ΔUs3gK-HA, gK-HA signals are strongly enhanced in the cytoplasm (forming large clusters), at the nuclear rim and in nuclei ( Figure 4B). At the ultrastructural level, Golgi membranes ( Figure 5), nuclear membranes and viral envelopes ( Figure 6) and the homogeneous structures in nuclei and cytoplasm (Figure 7) were distinctly labeled in CK177ΔUs3gK-HA infected cells. Immunogold labeling agrees with the distribution of gK-HA visualized by immunofluorescence implying that gK was, in addition to Golgi localization, translocated into nuclei, incorporated into nuclear membranes, and became part of the viral envelope during budding.   . CK177gK-HA infected cells show fine but dense distribution of gK-HA over the entire cytoplasm, at the nuclear rim and, at a less extent, in the nucleus. Distribution of Us3 is similar though more intense at the nuclear rim. In the absence of Us3, gK-HA signals are markedly enhanced in all three compartments. gK-HA partly localizes with wheat germ agglutinin (WGA) as a marker for Golgi membranes. The fate of the Golgi complex is under investigation using specific markers.

Discussion
The precise functions of Us3 kinase has been reviewed by Kato & Kawaguchi (2018). However, the mechanism of regulation phospholipid-biosynthesis is unknown (Wild et al., 2012). Membrane enlargement in the absence of Us3 is associated with structural alterations including folding of nuclear membranes and malformation of Golgi stacks, respectively, and insertion of proteins into nuclear and Golgi membranes. Immunolabeling using antibodies against the HA tag recognized gK to be one component of altered membranes suggesting that deposition/ insertion of gK is related to membrane overproduction in the absence of Us3.
HSV-1ΔUs3 virions are infective even though they are not released from the PNS, and do not pass the Golgi complex. gK has been reported to be involved in viral envelopment by Golgi membranes, not by nuclear membranes (Melancon et al., 2005). gK is essential for infectivity playing a significant role in viral entry (Foster et al., 2001;Musarrat et al., 2018). Therefore, gK must be provided during budding of HSV-1ΔUs3 at nuclear membranes. The intense labeling of gK on nuclear membranes and viral envelopes clearly demonstrates that gK becomes part of the viral envelope during budding of HSV-1ΔUs3. gK was also found in nuclei in both HSV-1ΔUs3 and CK177gK-HA infected cells indicating that gK is transported into the nucleus in the presence or absence of Us3. Indeed, others have also observed gK incorporated into nuclear membranes in the context of wtHSV-1 infection (Rajcani & Kudelova, 1998) suggesting that gK may also play a significant role in envelopment of wtHSV-1 at nuclear membranes.

Conclusion
Without Us3, Golgi and nuclear membranes proliferate in association with incorporation of gK that also localizes on virions remaining stuck in the PNS but mature to infectivity suggesting that i) Us3 kinase is involved in regulation in nuclear and Golgi membranes assembly possibly in association with gK synthesis and/or distribution, ii) gK may be the target of Us3 phosphorylation, which is needed for virions to proceed out of the PNS, and iii) gK may also be involved in nucleus-tocytoplasm translocation in wtHS-1 because gK localizes on nuclei and nuclear rim in the presence of Us3. We hypothesize that Us3 interplays with mechanisms regulating synthesis and arrangement of Golgi membranes, nuclear membranes and gK, which might be the most significant role of Us3 remaining to be investigated.

Grant information
This study was supported by the Foundation for Scientific Research at the University of Zürich, Switzerland.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed. Competing Interests: Reviewer Expertise: Molecular virology and immunopathogenesis of herpes viruses I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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