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. 2012 Jan;86(1):473-83.
doi: 10.1128/JVI.05981-11. Epub 2011 Oct 19.

Analysis of the interaction between the essential herpes simplex virus 1 tegument proteins VP16 and VP1/2

Stanislava Svobodova  1 , Susanne Bell, Colin M Crump
Affiliations

Analysis of the interaction between the essential herpes simplex virus 1 tegument proteins VP16 and VP1/2

Stanislava Svobodova et al. J Virol. 2012 Jan.

Abstract

The incorporation of tegument proteins into the herpes simplex virus 1 (HSV-1) virion during virion assembly is thought to be a complex, multistage process occurring via numerous interactions between the tegument and the capsid, within the tegument, and between the tegument and the envelope. Here, we set out to examine if the direct interaction between two essential tegument proteins VP1/2 and VP16 is required for connecting the inner tegument with the outer tegument. By using glutathione S-transferase (GST) pulldowns, we identified an essential role of lysine 343 in VP16, mutation of which to a neutral amino acid abrogated the interaction between VP1/2 and VP16. When the K343A substitution was inserted into the gene encoding VP16 (UL48) of the viral genome, HSV-1 replicated successfully although its growth was delayed, and final titers were reduced compared to titers of wild-type virus. Surprisingly, the mutated VP16 was incorporated into virions at levels similar to those of wild-type VP16. However, the analysis of VP16 on cytoplasmic capsids by fluorescence microscopy showed that VP16 associated with cytoplasmic capsids less efficiently when the VP16-VP1/2 interaction was inhibited. This implies that the direct interaction between VP1/2 and VP16 is important for the efficiency/timing of viral assembly but is not essential for HSV-1 replication in cell culture. These data also support the notion that the incorporation of tegument proteins into the herpesviruses is a very complex process with significant redundancy.

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Figures

Fig 1
Fig 1
Mapping of the VP16 region involved in VP1/2 binding. (A) A schematic representation of GST-tagged VP16 truncation mutants and summary of results, indicating the GST-VP16 truncation mutants that bound very strongly (++++), strongly (+++), moderately (++), or weakly (+) or did not bind (−) to GFP-VP1/2NT. See Materials and Methods for details of quantification. FL, full-length. (B and C) 293T cells were cotransfected with GFP-VP1/2NT and the indicated GST-VP16 truncation mutants. Cell lysates were harvested at 48 h posttransfection and incubated with glutathione-Sepharose beads. Both cell lysates and bound protein complexes (GST pulldowns) were separated by SDS-PAGE and analyzed by Western blotting with anti-GST and anti-GFP antibodies. α, anti.
Fig 2
Fig 2
Alanine scanning mutagenesis of VP16. (A) Amino acid sequence of the region spanning residues 332 to 349 of VP16 and mutants (M1 to M6) created in the context of GST-VP16(1–411). (B to G) 293T cells were cotransfected with GFP-VP1/2NT and indicated GST-VP16(1–411) mutants. Cell lysates were harvested at 48 h posttransfection and incubated with glutathione-Sepharose beads. Both cell lysates and bound protein complexes were separated by SDS-PAGE and analyzed by Western blotting with anti-GST and anti-GFP antibodies.
Fig 3
Fig 3
K343A substitution in VP16 inhibits interaction with VP1/2 during infection. (A and C) HaCaT cells were infected with the indicated viruses, and cell lysates were harvested at 18 hpi. (B and D) Cell lysates were subjected to immunoprecipitation (IP) using an anti-VP16 (αVP16) antibody. Cell lysates (A and C) and bound protein complexes (B and D) were separated by SDS-PAGE and analyzed by Western blotting using the indicated antibodies. Tubulin and the heavy chain of the anti-VP16 antibody were included as loading controls.
Fig 4
Fig 4
K343A mutation in VP16 moderately inhibits virus replication. HaCaT cells were infected with the indicated viruses and harvested at various time points. (A and B) Total infectious virus yields were determined by plaque assay on Vero cells. The titers represent mean PFU/ml, and error bars represent standard errors of the means of triplicate samples. (C and D) Cell lysates were separated by SDS-PAGE and analyzed by Western blotting using the indicated antibodies. Tubulin was included as a loading control.
Fig 5
Fig 5
VP16(K343A) is incorporated into virions in amounts similar to those of wt VP16. Extracellular virions were purified from the medium of infected HaCaT cells by Ficoll density gradient centrifugation, and VP5 levels in a range of PFU concentrations were analyzed by Western blotting. The appropriate PFU amounts to give equivalent levels of VP5 for each purified virion preparation were separated by SDS-PAGE and analyzed by Western blotting using the indicated antibodies. Numbers on the left represent molecular weight markers.
Fig 6
Fig 6
Analysis of VP16-Ch and VP16(K343A)-Ch association with fluorescent capsids. Monolayers of HFF-hTERT cells grown on coverslips were infected with HSV-VP16-Ch/VP26-Y (A) or HSV-VP16(K343A)-Ch/VP26-Y (B). Cells were fixed and analyzed by fluorescence microscopy. Representative cells on right are shown with higher magnification (boxed area) slown along the right side of each large image. Scale bar of insets, 1 μm. Histograms at left show red fluorescence intensities of cytoplasmic EYFP-labeled particles.
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