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Herpes simplex virus-1 (HSV-1), like all herpesviruses, is a large complex DNA virus containing up to 16 different viral membrane proteins in its envelope. Illustration and Flash animations byKarin ChristensenScientific, Medical and Veterinary Illustration.
Table 1 below shows a list of the HSV-1 genes together with information about the encoded protein. IntroductionThe correct sub-cellular localization of viral envelope proteins is crucial for the assembly of enveloped viruses. Antibody Internalization AssaysCOS-7 cells grown on glass coverslips were transfected using Trans-IT LT1 according to the manufacturer’s instructions (Mirus Bio, Madison, WI, USA).
This is computed by comparing the amino acid sequences of protein homologs in the HSV-1 and HSV-2 genomes. To ensure incorporation of all viral membrane proteins into the envelope, they need to be localized to the appropriate intracellular membranes either via the endocytic pathway or by direct targeting to assembly sites from the biosynthetic secretory pathway.
The cells were lysed to release cell-associated virus by sonication for 20 s at 39% amplitude in a cup horn sonicator (Fisher Scientific) and virus stocks were stored at ?80 °C. In Vero cells, the deletion of gM from the virus resulted in peak titers similar to WT, with a c.a. Total infectious virus yields were determined by plaque assay using the appropriate complementing cells lines. This revealed that gM and pUL20 were not expressed in cells infected with the appropriate viruses (Figure 3e).
The localization of cellular membrane proteins frequently involves discrete targeting motifs, short sequences present in cytoplasmic domains of such membrane proteins that mediate their inclusion into transport vesicles via direct or indirect interactions with the vesicle forming coat machinery. Viral titers were determined by plaque assays on Vero cells (*) or on the appropriate complementing cell line (**). Unfortunately no antibodies that detect gK by Western blotting were available for these studies.
The best-understood class of cellular targeting motifs is the so-called tyrosine based motifs (YXX?, where X indicates any amino acid and ? indicates an amino acid with a bulky hydrophobic side chain), which interact with clathrin adaptor complexes [1].Herpesviruses are large and structurally complex DNA viruses that infect a wide variety of vertebrates and cause many clinical and veterinary problems. Cells were then lysed in their media by three cycles of freeze thawing and total virus titers were determined by plaque assays. All viruses were constructed using a bacterial artificial chromosome (BAC)-cloned HSV-1 genome (strain KOS; [26]) using the two-step Red-mediated recombination method [27].
The replication of ?gK and ?pUL20 however were severely attenuated, showing approximately 450- and 2000-fold reduction in final titers compared to WT respectively (Figure 3a,b).

Molecular mass markers (in kDa) are indicated on the right; (f) Plaque formation of the indicated viruses was determined by standard plaque assay on Vero, VK302 and Fd20-1 cells at 3 days post infection. However, we noted that deletion of gK resulted in reduction of pUL20 levels, consistent with previously published results that the two form a complex and are reliant on one another for processing [10,11].
The envelope of herpesviruses includes many different viral envelope proteins, for example the alphaherpesvirus herpes simplex virus-1 (HSV-1) envelope contains at least 12, and potentially up to 16 different virally encoded transmembrane proteins [2]. The samples were then incubated with Alexa-647 conjugated anti-mouse secondary antibody (Life Technologies, Grand Island, NY, USA). All recombiant virus BAC-clones were carefully analysed by restriction enzyme digest to ensure correct recombination had occurred and no detectable deletions or insertions were present.
US4 and US5) encoded in the S segment and proteins involved in control of transcription such as RL1, RL2 and US1. HSV-1 envelope proteins mediate a wide range of roles during the virus life cycle and are, thus, important components of the virion. The coverslips were then washed and mounted onto glass slides using ProLong Gold antifade reagent containing DAPI (Life Technologies).
All mutations were verified by sequencing PCR fragments covering the respective regions of the genome that were generated from infected cell DNA.
The most conserved genes are those involved in DNA encapsidation, DNA replication and capsid structure. All mutations were present as expected with the exception of the singly mutated KOS ?pUL20, which had also acquired an additional base insertion of a single G in the first stop codon, leading to a F20W mutation as well as a frameshift rather than the designed stop codons (Figure 2c).
However the replication of the deletion viruses was not rescued to WT levels indicating that the complementation provided by the cell lines is not complete, although we cannot formally rule out the possibility that random additional changes may have occurred during the construction of the recombinant viruses that are not fully rescued by these complementing cell lines.
The resulting translation product from this mutated gene would contain the first 19 amino acids of pUL20, followed by 23 amino acids from the out of frame sequence before a stop codon is reached.
In the VK302 and Fd20-1 cell lines, the deletion of gM results in similar final titers compared to WT (Figure 3c,d).
Endocytic targeting motifs have been characterized in gB and gE, which allow for trafficking to the cell surface and subsequent internalization where they accumulates intracellularly [4,5]. Samples were then incubated with anti-VP5 antibody (Table 2) followed by subtype specific secondary antibodies (anti-IgG2a-Alexa-488 and anti-IgG2b-Alexa-568; (Life Technologies) and mounting on slides. Such a drastically different protein sequence would not be expected to retain any normal pUL20 functionality. Fluorescence microscopy images were captured using a 60 ? 1.4-numerical-aperture oil objective (Olympus, Tokyo, Japan) of an Olympus IX81 inverted wide field microscope using Image-Pro Plus software.

In HSV-1, the 5' end of the gK-encoding gene (UL53) overlaps by 45 bp with the 3' end of the essential DNA synthesis gene UL52 (helicase-primase subunit). The severe attenuation in single-step growth curves observed with both gK- and pUL20-deficient viruses in non-complementing cell lines could be the result of reduced numbers of virions being produced, the production of non-infectious virions, or a combination of the two. The mutations introduced into the gK-encoding gene were therefore designed downstream of the UL52 stop codon so as to not affect its expression. Virus particles were then pelleted by centrifugation (48,600? g, 3 h, 4 °C, Beckman TLA-100.3). Likewise the 5' end of the gM-encoding gene (UL10) overlaps with the 5' end of the essential origin binding protein gene (UL9), which is in the opposite orientation to UL10, by 55 base pairs, and so the stop codons were introduced into UL10 at 100bp 5' to the UL9 start codon to avoid affecting UL9 expression.
The pellet was resuspended in 50 µL of SDS-PAGE sample buffer and analysed by Western blotting with various antibodies (Table 2). Therefore, viruses lacking gK or pUL20 require complementing cell lines for propagation of virus stocks (Table 3).
Studies have demonstrated that HSV-1 gK and pUL20 form a complex, and the correct intracellular trafficking, localization and function of these proteins relies on their co-expression [10,11].
Mutant viruses obtained from these Vero-based complementing cell lines are fully infectious in complementing cells. Infection of non-complementing cell lines can be used to investigate the effects of these gene deletions on post-entry stages of a single round of viral replication. Structurally, gK has been described to have an N-terminal signal sequence and either four transmembrane domains with extracellularly located N- and C-termini [17,18] or three transmembrane domains with the C-terminal tail located intracellularly [21,22].
As a control for equivalent levels of infection, cells were then permeabilised and incubated with a VP5-specific antibody, followed by subtype specific secondary antibodies. VP5 (the major capsid protein) is a late gene product and localizes primarily to the nucleus. Only cells that demonstrated equivalent levels of VP5 were imaged, and the same exposure settings and post-acquisition processing was used for all samples.

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