Physical and Functional Interactions between the Herpes Simplex Virus UL15 and UL28 DNA Cleavage and Packaging Proteins

Herpes simplex virus (HSV) DNA is cleaved from concatemers and packaged into capsids in infected cell nuclei. This process requires seven viral proteins, including UL15 and UL28. UL15 expressed alone displays a nuclear localization, while UL28 remains cytoplasmic. Coexpression with UL15 enables UL28 to enter nuclei, suggesting an interaction between the two proteins. Additionally, UL28 copurified with UL15 from HSV-infected cells after ion-exchange and DNA affinity chromatography, and the complex sedimented as a 1:1 heterodimer upon sucrose gradient centrifugation. These findings are evidence of a physical interaction of UL15 and UL28 and a functional role for UL15 in directing UL28 to the nucleus.     

Identification of an essential domain in the herpes simplex virus 1 UL34 protein that is necessary and sufficient to interact with UL31 protein

Previous results have indicated that the herpes simplex virus 1 UL31 and UL34 proteins interact and form a complex at the inner nuclear membranes of infected cells, where both play important roles in the envelopment of nucleocapsids at the inner nuclear membrane. In the work described here, mapping studies using glutathione S-transferase pull-down assays indicated that amino acids 137 to 181 of the UL34 protein are sufficient to mediate an interaction with the UL31 protein. A recombinant virus (v3480) lacking UL34 codons 138 to 181 was constructed. Similar to a UL34 null virus, v3480 failed to replicate on Vero cells and grew to a limited extent on rabbit skin cells. A UL34-expressing cell line restored v3480 growth and plaque formation. Similar to the localization of UL31 protein in cells infected with a UL34 null virus, the UL31 protein was present in the nuclei of Hep2 cells infected with v3480. Hep2 cells infected with v3480 contained the UL34 protein in the cytoplasm, the nucleus, and the nuclear membrane, and this was noted to be similar to the appearance of cells infected with a UL31 null virus. In transient expression assays, the interaction between UL34 amino acids 137 to 181 and the UL31 protein was sufficiently robust to target green fluorescent protein and emerin to intranuclear sites that contained the UL31 protein. These data indicate that amino acids 137 to 181 of the UL34 protein are (i) sufficient to mediate interactions with the UL31 protein in vitro and in vivo, (ii) necessary for the colocalization of UL31 and UL34 in infected cells, and (iii) essential for normal viral replication.     

Herpes simplex virus type 1 portal protein UL6 interacts with the putative terminase subunits UL15 and UL28

The herpes simplex virus type 1 (HSV-1) UL6, UL15, and UL28 proteins are essential for cleavage of replicated concatemeric viral DNA into unit length genomes and their packaging into a preformed icosahedral capsid known as the procapsid. The capsid-associated UL6 DNA-packaging protein is located at a single vertex and is thought to form the portal through which the genome enters the procapsid. The UL15 protein interacts with the UL28 protein, and both are strong candidates for subunits of the viral terminase, a key component of the molecular motor that drives the DNA into the capsid. To investigate the association of the UL6 protein with the UL15 and UL28 proteins, the three proteins were produced in large amounts in insect cells with the baculovirus expression system. Interactions between UL6 and UL28 and between UL6 and UL15 were identified by an immunoprecipitation assay. These results were confirmed by transiently expressing wild-type and mutant proteins in mammalian cells and monitoring their distribution by immunofluorescence. In cells expressing the single proteins, UL6 and UL15 were concentrated in the nuclei whereas UL28 was found in the cytoplasm. When the UL6 and UL28 proteins were coexpressed, UL28 was redistributed to the nuclei, where it colocalized with UL6. In cells producing either of two cytoplasmic UL6 mutant proteins and a functional epitope-tagged form of UL15, the UL15 protein was concentrated with the mutant UL6 protein in the cytoplasm. These observed interactions of UL6 with UL15 and UL28 are likely to be of major importance in establishing a functional DNA-packaging complex at the portal vertex of the HSV-1 capsid.     

Linker insertion mutations in the herpes simplex virus type 1 UL28 gene: effects on UL28 interaction with UL15 and UL33 and identification of a second-site mutation in the UL15 gene that suppresses a lethal UL28 mutation

The UL28 protein of herpes simplex virus type 1 (HSV-1) is one of seven viral proteins required for the cleavage and packaging of viral DNA. Previous results indicated that UL28 interacts with UL15 and UL33 to form a protein complex (terminase) that is presumed to cleave concatemeric DNA into genome lengths. In order to define the functional domains of UL28 that are important for DNA cleavage/packaging, we constructed a series of HSV-1 mutants with linker insertion and nonsense mutations in UL28. Insertions that blocked DNA cleavage and packaging were found to be located in two regions of UL28: the first between amino acids 200 to 400 and the second between amino acids 600 to 740. Insertions located in the N terminus or in a region located between amino acids 400 and 600 did not affect virus replication. Insertions in the carboxyl terminus of the UL28 protein were found to interfere with the interaction of UL28 with UL33. In contrast, all of the UL28 insertion mutants were found to interact with UL15 but the interaction was reduced with mutants that failed to react with UL33. Together, these observations were consistent with previous conclusions that UL15 and UL33 interact directly with UL28 but interact only indirectly with each other. Revertant viruses that formed plaques on Vero cells were detected for one of the lethal UL28 insertion mutants. DNA sequence analysis, in combination with genetic complementation assays, demonstrated that a second-site mutation in the UL15 gene restored the ability of the revertant to cleave and package viral DNA. The isolation of an intergenic suppressor mutant provides direct genetic evidence of an association between the UL28 and UL15 proteins and demonstrates that this association is essential for DNA cleavage and packaging.     

Putative terminase subunits of herpes simplex virus 1 form a complex in the cytoplasm and interact with portal protein in the nucleus

Herpes simplex virus (HSV) terminase is an essential component of the molecular motor that translocates DNA through the portal vertex in the capsid during DNA packaging. The HSV terminase is believed to consist of the UL15, UL28, and UL33 gene products (pUL15, pUL28, and pUL33, respectively), whereas the HSV type 1 portal vertex is encoded by UL6. Immunoprecipitation reactions revealed that pUL15, pUL28, and pUL33 interact in cytoplasmic and nuclear lysates. Deletion of a canonical nuclear localization signal (NLS) from pUL15 generated a dominant-negative protein that, when expressed in an engineered cell line, decreased the replication of wild-type virus up to 80-fold. When engineered into the genome of recombinant HSV, this mutation did not interfere with the coimmunoprecipitation of pUL15, pUL28, and pUL33 from cytoplasmic lysates of infected cells but prevented viral replication, most nuclear import of both pUL15 and pUL28, and coimmunoprecipitation of pUL15, pUL28, and pUL33 from nuclear lysates. When the pUL15/pUL28 interaction was reduced in infected cells by the truncation of the C terminus of pUL28, pUL28 remained in the cytoplasm. Whether putative terminase components localized in the nucleus or cytoplasm, pUL6 localized in infected cell nuclei, as viewed by indirect immunofluorescence. The finding that the portal and terminase do eventually interact was supported by the observation that pUL6 coimmunoprecipitated strongly with pUL15 and weakly with pUL28 from extracts of infected cells in 1.0 M NaCl. These data are consistent with the hypothesis that the pUL15/pUL28/pUL33 complex forms in the cytoplasm and that an NLS in pUL15 is used to import the complex into the nucleus where at least pUL15 and pUL28 interact with the portal to mediate DNA packaging.     

Identification of nuclear and nucleolar localization signals of pseudorabies virus (PRV) early protein UL54 reveals that its nuclear targeting is required for efficient production of PRV

The pseudorabies virus (PRV) early protein UL54 is a homologue of herpes simplex virus 1 (HSV-1) immediate-early protein ICP27, which is a multifunctional protein that is essential for HSV-1 infection. In this study, the subcellular localization and nuclear import signals of PRV UL54 were characterized. UL54 was shown to predominantly localize to the nucleolus in transfected cells. By constructing a series of mutants, a functional nuclear localization signal (NLS) and a genuine nucleolar localization signal (NoLS) of UL54 were for the first time identified and mapped to amino acids (61)RQRRR(65) and (45)RRRRGGRGGRAAR(57), respectively. Additionally, three recombinant viruses with mutations of the NLS and/or the NoLS in UL54 were constructed based on PRV bacterial artificial chromosome (BAC) pBecker2 to test the effect of UL54 nuclear targeting on viral replication. In comparison with the wild-type virus, a recombinant virus harboring an NLS or NoLS mutation of UL54 reduced viral production to different extents. However, mutations of both the NLS and NoLS targeted UL54 to the cytoplasm in recombinant virus-infected cells and significantly impaired viral replication, comparable to the UL54-null virus. In addition, a virus lacking the NLS or the NoLS displayed modest defects in viral gene expression and DNA synthesis. However, deletion of both the NLS and the NoLS resulted in severe defects in viral gene expression and DNA synthesis, as well as production of infectious progeny. Thus, we have identified a classical NLS and a genuine NoLS in UL54 and demonstrate that the nuclear targeting of UL54 is required for efficient production of PRV.     

Direct and specific binding of the UL16 tegument protein of herpes simplex virus to the cytoplasmic tail of glycoprotein E

The UL16 tegument protein of herpes simplex virus (HSV) is conserved throughout all of the herpesvirus families. Previous studies have shown that the binding of HSV to heparan sulfate molecules on the host cell triggers the release of UL16 from the capsid, but the mechanism by which the signal is sent from the virion surface into the tegument is unknown. Here, we report that a glutathione S-transferase chimera bearing the cytoplasmic tail of viral glycoprotein E (gE) is capable of binding to UL16 in lysates of eukaryotic cells or purified from bacteria. Moreover, mass spectrometry studies of native-UL16 complexes purified from infected cells also revealed the presence of gE. Proof that UL16-gE can interact within cells required the fortuitous discovery of a mutant possessing only the first 155 residues of UL16. Confocal microscopy of cotransfected cells revealed that this mutant colocalized with gE in the cytoplasm, whereas it was found throughout the cytoplasm and nucleus when expressed alone. In contrast, the full-length UL16 molecule was very poorly capable of finding gE. Moreover, membrane flotation assays showed that UL16(1-155) was able to float to the top of sucrose step gradients when coexpressed with gE, whereas full-length UL16 was not. Thus, the discovery of the UL16(1-155) mutant confirmed the specific in vitro interaction with gE and provides evidence that a binding domain at the N terminus of UL16 may be controlled by a regulatory domain within the C terminus. These findings suggest the possibility that the UL16-gE interaction may play roles in the tegument signaling mechanism, virus budding, and the gE-mediated mechanism of cell-to-cell spread.     

A Mutation Deleting Sequences Encoding the Amino Terminus of Human Cytomegalovirus UL84 Impairs Interaction with UL44 and Capsid Localization

Protein-protein interactions are required for many biological functions. Previous work has demonstrated an interaction between the human cytomegalovirus DNA polymerase subunit UL44 and the viral replication factor UL84. In this study, glutathione S-transferase pulldown assays indicated that residues 1 to 68 of UL84 are both necessary and sufficient for efficient interaction of UL84 with UL44 in vitro. We created a mutant virus in which sequences encoding these residues were deleted. This mutant displayed decreased virus replication compared to wild-type virus. Immunoprecipitation assays showed that the mutation decreased but did not abrogate association of UL84 with UL44 in infected cell lysate, suggesting that the association in the infected cell can involve other protein-protein interactions. Further immunoprecipitation assays indicated that IRS1, TRS1, and nucleolin are candidates for such interactions in infected cells. Quantitative real-time PCR analysis of viral DNA indicated that the absence of the UL84 amino terminus does not notably affect viral DNA synthesis. Western blotting experiments and pulse labeling of infected cells with [(35)S]methionine demonstrated a rather modest downregulation of levels of multiple proteins and particularly decreased levels of the minor capsid protein UL85. Electron microscopy demonstrated that viral capsids assemble but are mislocalized in nuclei of cells infected with the mutant virus, with fewer cytoplasmic capsids detected. In sum, deletion of the sequences encoding the amino terminus of UL84 affects interaction with UL44 and virus replication unexpectedly, not viral DNA synthesis. Mislocalization of viral capsids in infected cell nuclei likely contributes to the observed decrease in virus replication.     

The pseudorabies virus homology of the herpes simplex virus UL21 gene product is a capsid protein which is involved in capsid maturation.

We mutagenized, mapped, and sequenced the pseudorabies virus (PRV) homology of gene UL21 of herpes simplex virus type 1. A polyclonal mouse antiserum against the protein encoded by the UL21 homolog was generated and used to monitor the expression and subcellular localization of the UL21-encoded protein. We found that the protein is identical to a previously detected PRV capsid protein. We analyzed viable PRV strains encoding mutant UL21 homologys, truncated by insertion of an oligonucleotide that contains stop codons in all reading frames. In two PRV mutants carrying the oligonucleotide at two sites within the gene, processing of newly replicated viral DNA was impaired. In addition, we show that one of the UL21 mutants has strongly reduced virulence for mice.     

Herpes Simplex Virus 1 Glycoprotein M and the Membrane-Associated Protein UL11 Are Required for Virus-Induced Cell Fusion and Efficient Virus Entry

Herpes simplex virus 1 (HSV-1) facilitates virus entry into cells and cell-to-cell spread by mediating fusion of the viral envelope with cellular membranes and fusion of adjacent cellular membranes. Although virus strains isolated from herpetic lesions cause limited cell fusion in cell culture, clinical herpetic lesions typically contain large syncytia, underscoring the importance of cell-to-cell fusion in virus spread in infected tissues. Certain mutations in glycoprotein B (gB), gK, UL20, and other viral genes drastically enhance virus-induced cell fusion in vitro and in vivo. Recent work has suggested that gB is the sole fusogenic glycoprotein, regulated by interactions with the viral glycoproteins gD, gH/gL, and gK, membrane protein UL20, and cellular receptors. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK. Virus-induced cell fusion caused by deletion of the carboxyl-terminal 28 amino acids of gB or the dominant syncytial mutation in gK (Ala to Val at amino acid 40) was drastically reduced in the absence of gM. Similarly, syncytial mutations in either gB or gK did not cause cell fusion in the absence of UL11. Neither the gM nor UL11 gene deletion substantially affected gB, gC, gD, gE, and gH glycoprotein synthesis and expression on infected cell surfaces. Two-way immunoprecipitation experiments revealed that the membrane protein UL20, which is found as a protein complex with gK, interacted with gM while gM did not interact with other viral glycoproteins. Viruses produced in the absence of gM or UL11 entered into cells more slowly than their parental wild-type virus strain. Collectively, these results indicate that gM and UL11 are required for efficient membrane fusion events during virus entry and virus spread.     

Identification of nuclear export signal in UL37 protein of herpes simplex virus type 2

The UL37 gene of herpes simplex virus (HSV) encodes a 120-kDa phosphoprotein associated with the virion. In this study, we have generated a rabbit polyclonal antiserum against HSV-2 UL37 protein, and examined its intracellular localization by immunofluorescence study. In infected cells, specific fluorescence was detectable in the perinuclear region. In transfected cells, UL37 protein was observed mainly in the cytoplasm. Transfection assays of deletion mutants of UL37 protein suggested that the leucine rich region (LRR) containing amino acids 263-273 may be important for cytoplasmic localization. Deletion of the LRR or substitution of the leucine residues resulted in nuclear remaining of UL37 protein. Moreover, the LRR could export green fluorescent protein (GFP) to the cytoplasm as a fusion protein and this export was blocked by leptomycin B treatment, indicating that the LRR acted as a nuclear export signal. These results suggest that UL37 protein fulfills a role as a shuttle between the nucleus and the cytoplasm through the LRR.     

Human cytomegalovirus UL84 localizes to the cell nucleus via a nuclear localization signal and is a component of viral replication compartments

The UL84 open reading frame encodes a protein that is required for origin-dependent DNA replication and interacts with the immediate-early protein IE2 in lytically infected cells. Transfection of UL84 expression constructs showed that UL84 localized to the nucleus of transfected cells in the absence of any other viral proteins and displayed a punctate speckled fluorescent staining pattern. Cotransfection of all the human cytomegalovirus replication proteins and oriLyt, along with pUL84-EGFP, showed that UL84 colocalized with UL44 (polymerase accessory protein) in replication compartments. Experiments using infected human fibroblasts demonstrated that UL84 also colocalized with UL44 and IE2 in viral replication compartments in infected cells. A nuclear localization signal was identified using plasmid constructs expressing truncation mutants of the UL84 protein in transient transfection assays. Transfection assays showed that UL84 failed to localize to the nucleus when 200 amino acids of the N terminus were deleted. Inspection of the UL84 amino acid sequence revealed a consensus putative nuclear localization signal between amino acids 160 and 171 (PEKKKEKQEKK) of the UL84 protein.     

Nucleocytoplasmic shuttling of bovine herpesvirus 1 UL47 protein in infected cells

Previous studies with transfected cells have shown that the herpes simplex virus type 1 (HSV-1) and bovine herpesvirus 1 (BHV-1) UL47 proteins shuttle between the nucleus and the cytoplasm. HSV-1 UL47 has also been shown to bind RNA. Here we examine the BHV-1 UL47 protein in infected cells using a green fluorescent protein-UL47-expressing virus. We show that UL47 is detected in the nucleus early in infection. We use fluorescence loss in photobleaching to show that nuclear UL47 undergoes rapid nucleocytoplasmic shuttling. Furthermore, we demonstrate that actinomycin D inhibits the reaccumulation of UL47 in the nuclei of infected cells. These results suggest that UL47 exhibits behavior similar to that of previously characterized RNA-transporting proteins.     

Subcellular localization of herpes simplex virus type 1 UL51 protein and role of palmitoylation in Golgi apparatus targeting

The herpes simplex virus type 1 (HSV-1) UL51 gene products are virion-associated phosphoproteins with apparent molecular masses of 27, 29, and 30 kDa in HSV-1-infected cells. In this study, we have investigated the intracellular localization and distribution of UL51 protein both in infected cells and in transfected cells expressing only UL51. We found that this protein colocalized closely with Golgi marker proteins such as the Golgi-58K protein and GM130 in transfected cells expressing only UL51. However, in infected cells, the UL51 protein localized to the juxtanuclear region but only partially colocalized with the Golgi maker proteins. Mutant protein analysis revealed that the N-terminal 15 amino acid residues of the UL51 protein sufficed for this Golgi localization property. The UL51 protein redistributed on addition of brefeldin A. This was prevented by pretreatment with 2-deoxyglucose and sodium azide, which results in ATP depletion, but not by pretreatment with NaF and AlCl(3), which activates heterotrimeric G proteins. Moreover, we found that palmitoylation of the UL51 protein through the N-terminal cysteine at position 9 was necessary for its Golgi localization. Protease digestion analysis suggested that the UL51 protein localized on the cytoplasmic face of the membrane in UL51-transfected cells, while in infected cells it localized mainly to the inside of cytoplasmic vesicles and/or the viral envelope. Transmission immunoelectron microscopy revealed an association of UL51 protein-specific labeling with cytoplasmic virions and also with some membranous structure. We infer from these observations that internalization of UL51 protein into the cytoplasmic vesicle and/or virion may occur in association with viral envelopment in HSV-infected cells.     

Role of the UL25 protein in herpes simplex virus DNA encapsidation

The herpes simplex virus protein UL25 attaches to the external vertices of herpes simplex virus type 1 capsids and is required for the stable packaging of viral DNA. To define regions of the protein important for viral replication and capsid attachment, the 580-amino-acid UL25 open reading frame was disrupted by transposon mutagenesis. The UL25 mutants were assayed for complementation of a UL25 deletion virus, and in vitro-synthesized protein was tested for binding to UL25-deficient capsids. Of the 11 mutants analyzed, 4 did not complement growth of the UL25 deletion mutant, and analysis of these and additional mutants in the capsid-binding assay demonstrated that UL25 amino acids 1 to 50 were sufficient for capsid binding. Several UL25 mutations were transferred into recombinant viruses to analyze the effect of the mutations on UL25 capsid binding and on DNA cleavage and packaging. Studies of these mutants demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids and that the C terminus is essential for DNA packaging and the production of infectious virus through its interactions with other viral packaging or tegument proteins. Analysis of viral DNA cleavage demonstrated that in the absence of a functional UL25 protein, aberrant cleavage takes place at the unique short end of the viral genome, resulting in truncated viral genomes that are not retained in capsids. Based on these observations, we propose a model where UL25 is required for the formation of DNA-containing capsids by acting to stabilize capsids that contain full-length viral genomes.     

A novel herpes simplex virus 1 gene, UL43.5, maps antisense to the UL43 gene and encodes a protein which colocalizes in nuclear structures with capsid proteins.

An open reading frame mapping antisense to the UL43 gene of herpes simplex virus 1 encodes a protein with an apparent Mr of 38,000. The protein was detected in wild-type-infected cells with rabbit monospecific polyclonal antibody directed against a fusion protein containing all of the sequences encoded by the open reading frame. The antibody did not react with mutants from which the open reading frame was deleted. Expression of this gene, designated UL43.5, was grossly decreased or abolished in infected cells incubated in medium containing inhibitory concentrations of phosphonoacetic acid, suggesting that it is regulated as a gamma gene. UL43.5 is dispensable in cell culture. UL43.5 protein colocalized with the major capsid protein (infected cell protein 5) and the capsid scaffolding proteins (infected cell protein 35) in nuclear structures situated at the periphery of the nucleus. The predicted amino acid sequence indicates that the UL43.5 protein is a highly hydrophilic protein. The colocalization of UL43.5 protein with capsid proteins in discrete nuclear structures suggests that the former may be involved in assembly of viral particles in an accessory role in cells in culture.     

Herpes Simplex Virus Type 1 Cleavage and Packaging Proteins UL15 and UL28 Are Associated with B but Not C Capsids during Packaging

At least seven viral genes encode proteins (UL6, UL15, UL17, UL25, UL28, UL32, and UL33) that are required for DNA cleavage and packaging of herpes simplex virus type 1 (HSV-1) DNA. Sequence analysis reveals that UL15 shares homology with gp17, the large catalytic subunit of the bacteriophage T4 terminase. Thus, UL15 may play a direct role in the cleavage of viral DNA replication intermediates into monomers. In this study, we asked whether UL15 and other cleavage and packaging proteins could be detected in capsids isolated from infected cells. Consistent with previous studies showing that UL6 and UL25 are minor protein constituents of the capsids, we detected these proteins in both B and C capsids. In contrast, the previously identified full-length version (81 kDa) of UL15 was found predominantly in B capsids and in much smaller amounts in C capsids. In addition, the UL28 protein was found predominantly in B but not C capsids in a distribution similar to that of the 81-kDa version of UL15. These results suggest that UL28 and the 81-kDa form of UL15 are transiently associated with capsid intermediates during the packaging process. Surprisingly, however, a previously unidentified 87-kDa form of UL15 was found in the B and C capsids and in virions. Analysis of cells infected with mutants individually lacking UL6, UL15, UL25, UL28, or UL32 demonstrates that the lack of one cleavage and packaging protein does not affect the expression of the others. Furthermore, this analysis, together with guanidine HCl extraction analysis of purified capsids, indicates that UL6, UL25, and UL28 are able to associate with B capsids in the absence of other DNA cleavage and packaging proteins. On the other hand, the two UL15-related proteins (81 and 87 kDa) do not associate efficiently with B capsids in cells infected with UL6 and UL28 mutants. These results suggest that the ability of the UL15-related proteins to bind to B capsids may be mediated through interactions with UL6 and UL28.     

The UL25 protein of pseudorabies virus associates with capsids and localizes to the nucleus and to microtubules

The UL25 gene of pseudorabies virus (PrV) can encode a protein of about 57 kDa which is well conserved among herpesviruses. The UL25 protein of herpes simplex virus type 1 is a capsid constituent involved in virus penetration and capsid maturation. To identify and characterize the UL25 gene product of PrV, polyclonal mouse anti-UL25 antibodies were raised to a bacterially expressed fusion protein. In immunoblotting and immunoprecipitation assays of PrV-infected cell lysates, these anti-UL25 antisera specifically recognized a protein of the expected size with late expression kinetics. This 57-kDa product was also present in purified virions and was found to be associated with all types of capsids. Synthesis of a protein migrating at the same size point was directed from the eukaryotic expression plasmid pCG-UL25. To determine the subcellular localization of UL25, immunofluorescence studies with anti-UL25 antisera were performed on Nonidet P-40-extracted COS-7 cells infected with PrV or transfected with pCG-UL25. In PrV-infected cells, newly synthesized UL25 is directed mainly to distinct nuclear compartments, whereas UL25 expressed in the absence of other viral proteins is distributed more uniformly in the nucleus and colocalizes also with microtubules. To study the fate of UL25 at very early stages of infection, immunofluorescence experiments were performed on invading PrV particles in the presence or absence of drugs that specifically depolymerize components of the cytoskeleton. We found that the incoming nucleocapsids colocalize with microtubules during their transport to the nucleus and that UL25 remains associated with nucleocapsids during this transport.