A comparison of the primary amino acid sequences between PRV Us9 and its homologs in VZV, HSV-1, EHV-1, and BHV-1 showed several conserved motifs and domains (Fig. ). All of the Us9 proteins contain a dileucine or tyrosine-based endocytic motif. Mutation of the dileucine motif in PRV Us9 had a modest impact on the ability of Us9 to be endocytosed from the plasma membrane of swine epithelial cells (7
) but did not have a noticeable role in PRV Us9-mediated anterograde transneuronal spread in vivo (8
). By contrast, mutation of two tyrosines and two serines within the acidic domain region of PRV Us9 had a dramatic impact on Us9 endocytosis from the surface of infected PK15 cells, anterograde transneuronal spread of infection in the rat brain, and axonal sorting in dissociated primary neuronal cultures (8
). Specifically, changing the two conserved tyrosines to alanines abrogates anterograde sorting of PRV structural proteins; mutation of the two serine residues, which comprise casein kinase II phosphorylation sites, affects the rate of axonal sorting and subsequent transneuronal spread (8
). As shown in Fig. , these tyrosine and serine residues are highly conserved among all Us9 homologs.
FIG. 1. Primary amino acid sequences and conserved domains of PRV, VZV, HSV-1, EHV-1, and BHV-1 Us9 proteins. The dileucine- and tyrosine-based endocytic sorting motifs are highlighted in green. A portion of the acidic cluster domains that contain key tyrosine (more ...)
Other prominent domains within PRV Us9 are a positively charged region, enriched in arginine residues, found immediately before a long hydrophobic stretch of amino acids that constitutes a transmembrane domain. Kyte-Doolittle hydropathy analysis of this region (Fig. ) shows the disparate charge distribution between these domains; values greater than zero represent amino acids that are hydrophilic while negative values are hydrophobic. It is known that positively charged/basic amino acid regions located before the transmembrane domain of integral membrane proteins establish protein topology in the lipid bilayer (often referred to as the “positive-inside” rule, i.e., transmembrane proteins are orientated in such a way that positively charged arginine and lysine residues directly preceding the transmembrane domain are located within the cytoplasm) (31
). Furthermore, in the case of TA type II membrane proteins, basic regions before or after the transmembrane domain can also impact whether the protein is posttranslationally inserted into the mitochondrial outer membrane or the endoplasmic reticulum (ER) (3
). Therefore, all of the Us9 proteins shown in Fig. are predicted to have a type II membrane topology, i.e., to be TA membrane proteins. Indeed, it has been shown previously that PRV Us9 and VZV Us9 are TA membrane proteins (5
To facilitate the comparison of PRV Us9 to its homologs, the Us9 ORFs of VZV, HSV-1, BHV-1, and EHV-1 were PCR amplified and cloned in-frame with EGFP (primers and PCR templates are given in Table ). The resulting plasmids, pHA1 (VZV Us9-EGFP), pCK113 (HSV-1 Us9-EGFP), pCK114 (EHV-1 Us9-EGFP), and pCK115 (BHV-1 Us9-EGFP), were subsequently transfected into PK15 cells to analyze their subcellular localization.
PRV and VZV Us9 localize predominantly to the Golgi complex during infection (7
). PRV Us9-EGFP is also targeted to the TGN in transfected cells, similar to its localization inside infected cells (7
). To compare the TGN localization of PRV Us9-EGFP to the that of the other Us9 homologs, we cotransfected PRV, VZV, HSV-1, EHV-1, and BHV-1 Us9-EGFP expression constructs with GalT-mRFP1 into PK15 cells (23
). GalT is a membrane-bound enzyme enriched in the trans
-Golgi cisternae and is commonly used as a marker for the TGN (32
). We found that all of the Us9-EGFP constructs compartmentalized with GalT-RFP (Fig. ), suggesting that the TGN is the primary site for steady-state localization of HSV-1, EHV-1, and BHV-1 Us9 proteins (consistent with the known localization of PRV and VZV Us9). Slight plasma membrane staining was also observed in all cells expressing Us9-EGFP, consistent with the idea that a small population of Us9 cycles between the plasma membrane and the TGN (7
FIG. 2. PRV, VZV, HSV-1, EHV-1, and BHV-1 Us9-EGFP fusion proteins colocalize with the TGN marker GalT-RFP. Us9-EGFP and GalT-mRFP1 mammalian expression constructs were cotransfected into PK15 cells and fixed at 30 h posttransfection. Optical sections of transfected (more ...)
Though it has been shown that PRV and VZV Us9 are type II, TA membrane proteins (with their short C termini exposed to the lumen/cell surface and their N termini in the cytosol) (5
), it remained unclear if HSV-1, EHV-1, and BHV-1 Us9 proteins were also type II integral membrane proteins. In fact, HSV-1 Us9 was originally described to be a tegument protein (22
). As shown in Fig. , each of the Us9 homologs is predicted to have a transmembrane domain and to share similar topologies. Therefore, we examined whether all of the PRV Us9 homologs were in fact type II membrane proteins. PK15 cells were transiently transfected with PRV, VZV, HSV-1, EHV-1, and BHV-1 Us9-EGFP expression constructs for 30 h and fixed with paraformaldehyde. We reasoned that if the Us9-EGFP homologs had a type II orientation in the membrane, the EGFP moiety would be present on the surface of transfected cells and exposed to labeling with GFP polyclonal antiserum. Figure illustrates this idea, with EGFP fused to the C terminus of Us9 and located on the extracellular side of the plasma membrane. Alternatively, if the proteins were in a type I orientation, GFP would not be exposed to the cell surface, and no staining would be observed in the absence of permeabilization. Figure shows PK15 cells transfected with pEGFP-N1, fixed with paraformaldehyde, stained with primary GFP and secondary Alexa Fluor 546 antibodies in nonpermeabilizing buffer, and imaged by confocal microscopy. Note that no “red” EGFP staining was present on the cell surface as soluble EGFP protein inside the cell is protected from anti-GFP antibody. When 0.5% saponin was added to the buffer, the plasma membrane was permeabilized, and anti-GFP antibodies were able to stain intracellular EGFP (Fig. ) (nuclear membrane was not solubilized). As controls for the type II topology of a membrane protein, PRV and VZV Us9-EGFPs were clearly labeled at the cell surface by GFP antiserum in nonpermeabilizing buffer (Fig. ). Us9-EGFP in the TGN remained green by direct fluorescence but was not stained by anti-GFP antibodies. All of the Us9-EGFP homologs showed similar patterns of red surface staining at the plasma membrane with internal green perinuclear staining (Fig. ). These data strongly support the notion that HSV-1, EHV-1, and BHV-1 Us9 are all type II membrane proteins (along with their previously characterized PRV and VZV Us9 counterparts).
FIG. 3. PRV Us9 and its homologs have a type II membrane topology. The schematic illustrates our model for Us9-EGFP homologs with a type II orientation in the membrane. The EGFP moiety will be present on the surface of transfected cells and exposed to labeling (more ...)
PRV Us9 is essential for the sorting of viral structural components into the axon of infected neurons (28
), a process that subsequently impacts the virus's ability to undergo anterograde, transneuronal spread in vivo (6
). We reasoned that since all of the Us9 homologs closely resembled PRV Us9 (i.e., were type II membrane proteins, contained critical sorting domains, and localized to the TGN), perhaps they would functionally compensate for the loss of PRV Us9 in axonal sorting and anterograde spread of infection. Thus, we constructed PRV Us9-null recombinant strains that expressed wild-type VZV, HSV-1, EHV-1, and BHV-1 Us9 proteins. These proteins were expressed ectopically as described previously for PRV 328, a virus strain that expresses wild-type PRV Us9 under the human CMV immediate-early promoter in the gG locus (26
). Figure contains a schematic of PRV strains expressing PRV Us9 (PRV 328), VZV Us9 (PRV 334), HSV-1 Us9 (PRV 335), EHV-1 Us9 (PRV 336), and BHV-1 Us9 (PRV 337). Western blot analysis using polyclonal antiserum against PRV Us9, VZV Us9, HSV-1 Us9, and BHV-1 Us9 confirmed the expression of these proteins from infected PK15 cell lysates collected at 6 hpi (Fig. ). We did not observe any cross-reactivity of the various Us9 antisera with nonconjugate Us9 homologs, consistent with the high similarity between Us9 domains and minimal primary amino acid identity (Fig. ). Because we did not have antiserum against EHV-1 Us9, its expression was confirmed by RT-PCR on mRNA harvested from PK15 cells infected with PRV 336 (Fig. ). Primer sets designed to amplify 500 bp of the EHV-1 Us9 ORF and EGFP ORF were used in the RT-PCR. Note that the EHV-1 primers were able to amplify cDNA from PRV 336-infected cells but not cDNA from cells infected with PRV 328 (negative control). The EGFP ORF was detected in both samples as it is present in the Us9 mRNA transcripts but is not translated into protein due to the introduction of a stop codon between the Us9 and EGFP ORFs (see Materials and Methods).
FIG. 4. Construction of PRV strains ectopically expressing Us9. (A) Schematic representation of the genomes of PRV 328 (PRV Us9), PRV 334 (VZV Us9), PRV 335 (HSV-1 Us9), PRV 336 (EHV-1 Us9), and PRV 337 (BHV-1 Us9) (adapted from Lyman et al. ). Note that (more ...)
Next, we assessed whether the Us9 homologs could functionally compensate for the loss of PRV Us9 in neuronal anterograde spread of infection. We employed a well-described, compartmentalized chamber system to assess the anterograde sorting and spread capabilities of virus strains in vitro (Fig. ) (11
). Briefly, dissociated SCG neurons dissected from embryonic rats are plated in compartment S and allowed to mature for 2 weeks in the presence of nerve growth factor. During this differentiation period, axons are guided between a series of grooves across the methocellulose compartment M to the neurite compartment N. PK15 indicator cells are then plated on the neurites in compartment N. Cell bodies in compartment S are infected, and virus particles sort into axons in a Us9-dependent manner (the primary infection is confined to compartment S via silicone vacuum grease and a methocellulose barrier). Thus, infectious virus particles spread into compartment N—in the anterograde direction—exclusively through axons emanating from neuronal cell bodies that contact PK15 cells.
FIG. 5. Compensation for the loss of PRV Us9 in anterograde, neuron-to-cell spread of infection. A schematized representation (schematic adapted from reference 26) of the compartmented chamber system is present at the top of the figure. Cell bodies in compartment (more ...)
PRV 328 (PRV Us9), PRV 161 (Us9-null), PRV 334 (VZV Us9), PRV 335 (HSV-1 Us9), PRV 336 (EHV-1 Us9), and PRV 337 (BHV-1 Us9) were used to infect compartment S of chambers at an MOI of 10; each virus strain was examined in quadruplicate (Fig. ). At 24 hpi, contents of both the S and N compartments were collected separately, and titers for PFU were determined on PK15 cells. PRV 328, our positive control, had a median titer of 3.7 × 105
PFU/ml in chamber S, with a median titer of 1.4 ×107
in chamber N (indicating efficient anterograde neuron-to-cell spread). PRV 161, a Us9-null mutant (6
), showed a robust infection in compartment S but was essentially dead for neuron-to-cell spread of infection, with three chambers showing 0 PFU in the compartment N. One chamber had a compartment N titer of 3.7 × 103
PFU/ml. We have observed before an occasional “blip” of Us9-null mutant virus in compartment N at 24 hpi and attribute it to a single spread event (26
). Nevertheless, compared to PRV 328, this small amount of infectious virus is negligible. By comparison, virus strains expressing VZV Us9 and HSV-1 Us9 replicated efficiently in compartment S but showed no ability to compensate for the loss of PRV Us9 in anterograde neuron-to-cell spread; i.e., no PFU were detected in compartment N. By comparison, PRV strains expressing EHV-1 Us9 and BHV-1 Us9 were indistinguishable from PRV 328; both Us9 proteins fully complemented the loss of PRV Us9 in axonal sorting and subsequent neuron-to-cell spread.