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. 2007 Apr;81(7):3097-108.
doi: 10.1128/JVI.02201-06. Epub 2007 Jan 10.

UL20 protein functions precede and are required for the UL11 functions of herpes simplex virus type 1 cytoplasmic virion envelopment

Preston A Fulmer  1 , Jeffrey M Melancon, Joel D Baines, Konstantin G Kousoulas
Affiliations

UL20 protein functions precede and are required for the UL11 functions of herpes simplex virus type 1 cytoplasmic virion envelopment

Preston A Fulmer et al. J Virol. 2007 Apr.

Abstract

Egress of herpes simplex virus type 1 (HSV-1) from the nucleus of the infected cell to extracellular spaces involves a number of distinct steps, including primary envelopment by budding into the perinuclear space, de-envelopment into the cytoplasm, cytoplasmic reenvelopment, and translocation of enveloped virions to extracellular spaces. UL20/gK-null viruses are blocked in cytoplasmic virion envelopment and egress, as indicated by an accumulation of unenveloped or partially enveloped capsids in the cytoplasm. Similarly, UL11-null mutants accumulate unenveloped capsids in the cytoplasm. To assess whether UL11 and UL20/gK function independently or synergistically in cytoplasmic envelopment, recombinant viruses having either the UL20 or UL11 gene deleted were generated. In addition, a recombinant virus containing a deletion of both UL20 and UL11 genes was constructed using the HSV-1(F) genome cloned into a bacterial artificial chromosome. Ultrastructural examination of virus-infected cells showed that both UL20- and UL11-null viruses accumulated unenveloped capsids in the cytoplasm. However, the morphology and distribution of the accumulated capsids appeared to be distinct, with the UL11-null virions forming aggregates of capsids having diffuse tegument-derived material and the UL20-null virus producing individual capsids in close juxtaposition to cytoplasmic membranes. The UL20/UL11 double-null virions appeared morphologically similar to the UL20-null viruses. Experiments on the kinetics of viral replication revealed that the UL20/UL11 double-null virus replicated in a manner similar to the UL20-null virus. Additional experiments revealed that transiently expressed UL11 localized to the trans-Golgi network (TGN) independently of either gK or UL20. Furthermore, virus infection with the UL11/UL20 double-null virus did not alter the TGN localization of transiently expressed UL11 or UL20 proteins, indicating that these proteins did not interact. Taken together, these results show that the intracellular transport and TGN localization of UL11 is independent of UL20/gK functions, and that UL20/gK are required and function prior to UL11 protein in virion cytoplasmic envelopment.

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Figures

FIG. 1.
FIG. 1.
Schematic of the strategy for the construction of pYEbac102 mutant BACs. (A) The top line represents the prototypic arrangement of the HSV-1 genome, with the unique long (UL) and unique short (US) regions flanked by the terminal repeat (TR) and internal repeat (IR) regions. (B) Shown are the expanded genomic regions of the UL11 and UL20 ORFs, the approximate locations of the genomic sites to which insertion of the marker genes was targeted, and the primers used in diagnostic PCR to confirm the presence of each mutation. (C) PCR fragments containing the kanamycin resistance or GFP-zeocin resistance gene cassette flanked by approximately 50 bp of viral sequences on both sides were used for targeted GET recombination in E. coli to construct pYEbac102 mutant BACs with insertion-deletion mutations in the UL11 and/or UL20 ORFs, respectively. The approximate locations of the primers used in amplification of each PCR fragment are also shown. CMV, cytomegalovirus.
FIG. 2.
FIG. 2.
PCR-based diagnostic analysis of pYEbac102ΔUL11 and pYEbac102ΔUL20 mutants. Oligonucleotide primers a and b (Table 1) were utilized to amplify DNA fragments containing the inserted Kan gene cassette. (i) Amplification with primers a and b produced the predicted 1,701-bp DNA fragment for the pYEbac102ΔUL11 and pYEbac102ΔUL11ΔUL20 genomes, consistent with the insertion of the Kan gene cassette, and the predicted 794-bp fragment for the pYEbac102 and pYEbac102ΔUL20 controls. (ii) Amplification with primers c and d (Table 1) produced the predicted 2,469-bp DNA fragment for the pYEbac102ΔUL20 and pYEbac102ΔUL11ΔUL20, consistent with insertion of the GZ gene cassette, and the 999-bp predicted DNA fragment for the pYEbac102 and pYEbac102ΔUL11ΔUL20 controls.
FIG. 3.
FIG. 3.
Plaque phenotypes of UL11-null, UL20-null, and UL11/UL20 double-null viruses under complementing and noncomplementing conditions. Vero cell monolayers were either mock transfected or transfected with plasmids expressing UL11, UL20, or both UL11 and UL20. Transfected cells were infected at 24 h posttransfection with the corresponding viruses, pYEbac102, pYEbac102ΔUL11, pYEbac102ΔUL20, and pYEbac102ΔUL11ΔUL20. Individual viral plaques were visualized at 24 h postinfection by immunohistochemistry.
FIG. 4.
FIG. 4.
Viral replication kinetics. A comparison of the viral replication characteristics of YEbac102 (•), YEbac102ΔUL20 (▪), YEbac102ΔUL11 (▴), and YEbac102ΔUL11ΔUL20 (○) on Vero cells is shown. One-step growth kinetics of infections virus production were calculated after infection at an MOI of 2 followed by incubation at 37°C. Error bars indicate standard deviations.
FIG. 5.
FIG. 5.
Ultrastructural morphologies of the YEbac102ΔUL11 (a), YEbac102ΔUL20 (b), and YEbac102ΔUL11ΔUL20 (c) viruses. Confluent cell monolayers were infected with the indicated virus at an MOI of 2, incubated for 24 h at 37°C, and prepared for transmission electron microscopy. Panels A, low magnification of an infected cell; panels B and C, higher magnifications of the cells shown in panels A. Nuclear (N), cytoplasmic (C), and extracellular (E) spaces are marked; bars show the relative magnification scale.
FIG. 5.
FIG. 5.
Ultrastructural morphologies of the YEbac102ΔUL11 (a), YEbac102ΔUL20 (b), and YEbac102ΔUL11ΔUL20 (c) viruses. Confluent cell monolayers were infected with the indicated virus at an MOI of 2, incubated for 24 h at 37°C, and prepared for transmission electron microscopy. Panels A, low magnification of an infected cell; panels B and C, higher magnifications of the cells shown in panels A. Nuclear (N), cytoplasmic (C), and extracellular (E) spaces are marked; bars show the relative magnification scale.
FIG. 6.
FIG. 6.
Digital images of confocal micrographs showing UL11 localization. Vero cell monolayers were transfected with the UL11-GFP-expressing plasmid. At 24 h posttransfection, cells were washed thoroughly, fixed, and stained with the anti-GFP antibody (A1) or with the Golgi specific marker TGN46 (A2) and fluorescence was visualized by confocal microscopy. A3, overlay. Magnification, ×63; zoom, ×2.
FIG. 7.
FIG. 7.
Comparison of UL11 intracellular localization in the presence or absence of gK/UL20. Vero cells were transfected with a combination of plasmids expressing UL11-GFP, UL20, and/or gK. At 24 h posttransfection, cells were washed thoroughly, fixed, and prepared for confocal microscopy after staining with the appropriate antibodies under the appropriate conditions. UL11 was stained with the anti-GFP antibody, UL20 with the anti-FLAG antibody, and gK with the anti-V5 antibody. Magnification, ×63; zoom ×2.
FIG. 8.
FIG. 8.
Intracellular localization of the UL11 and UL20 proteins in virus-infected cells. Vero cells were transfected with plasmids expressing UL11-GFP, UL20, or both. At 24 h posttransfection, cells were infected at an MOI of 2 with YEbac102ΔUL11ΔUL20. At 24 h postinfection, cells were washed thoroughly, fixed, stained with appropriate antibodies, and prepared for confocal microscopy. Magnification, ×63; zoom ×2.
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