Hsp90 is involved in pseudorabies virus virion assembly via stabilizing major capsid protein VP5.

Many viruses utilize molecular chaperone heat shock protein 90 (Hsp90) for protein folding and stabilization, however, the role of Hsp90 in herpesvirus lifecycle is obscure. Here, we provide evidence that Hsp90 participates in pseudorabies virus (PRV) replication. Viral growth kinetics assays show that Hsp90 inhibitor geldanamycin (GA) abrogates PRV replication at the post-penetration step. Transmission electron microscopy demonstrates that dysfunction of Hsp90 diminishes the quantity of PRV nucleocapsids. Overexpression and knockdown of Hsp90 suggest that de novo Hsp90 is involved in PRV replication. Mechanismly, dysfunction of Hsp90 inhibits PRV major capsid protein VP5 expression. Co-immunoprecipitation and indirect immunofluorescence assays indicate that Hsp90 interacts with VP5. Interestingly, Hsp70, a collaborator of Hsp90, also interacts with VP5, but doesn't affect PRV growth. Finally, inhibition of Hsp90 results in PRV VP5 degradation in a proteasome-dependent manner. Collectively, our data suggest that Hsp90 contributes to PRV virion assembly and replication via stabilization of VP5.

Single Virus Tracking with Quantum Dots Packaged into Enveloped Viruses Using CRISPR.

Labeling viruses with high-photoluminescence quantum dots (QDs) for single virus tracking provides a visual tool to aid our understanding of viral infection mechanisms. However, efficiently labeling internal viral components without modifying the viral envelope and capsid remains a challenge, and existing strategies are not applicable to most viruses. Here, we have devised a strategy using the clustered regularly interspaced short palindromic repeats (CRISPR) imaging system to label the nucleic acids of Pseudorabies virus (PRV) with QDs. In this strategy, QDs were conjugated to viral nucleic acids with the help of nuclease-deactivated Cas9/gRNA complexes in the nuclei of living cells and then packaged into PRV during virion assembly. The processes of PRV-QD adsorption, cytoplasmic transport along microtubules, and nuclear entry were monitored in real time in both Vero and HeLa cells, demonstrating the utility and efficiency of the strategy in the study of viral infection.

Dissecting the Herpesvirus Architecture by Targeted Proteolysis.

Herpesvirus particles have a complex architecture consisting of an icosahedral capsid that is surrounded by a lipid envelope. Connecting these two components is a layer of tegument that consists of various amounts of 20 or more proteins. The arrangement of proteins within the tegument cannot easily be assessed and instead is inferred from tegument interactions identified in reductionist models. To better understand the tegument architecture, we have developed an approach to probe capsid-tegument interactions of extracellular viral particles by encoding tobacco etch virus (TEV) protease sites in viral structural proteins, along with distinct fluorescent tags in capsid and tegument components. In this study, TEV sites were engineered within the pUL36 large tegument protein, a critical structural element that is anchored directly on the capsid surface. Purified pseudorabies virus extracellular particles were permeabilized, and TEV protease was added to selectively cleave the exposed pUL36 backbone. Interactions with the capsid were assessed in situ by monitoring the fate of the fluorescent signals following cleavage. Although several regions of pUL36 are proposed to bind capsids, pUL36 was found stably anchored to the capsid exclusively at its carboxyl terminus. Two additional tegument proteins, pUL37 and pUS3, were tethered to the capsid via pUL36, whereas the pUL16, pUL47, pUL48, and pUL49 tegument proteins were not stably bound to the capsid.IMPORTANCE Neuroinvasive alphaherpesviruses produce diseases of clinical and economic significance in humans and veterinary animals but are predominantly associated with less serious recurrent disease. Like all viruses, herpesviruses assemble a metastable particle that selectively dismantles during initial infection. This process is made more complex by the presence of a tegument layer that resides between the capsid surface and envelope. Components of the tegument are essential for particle assembly and also serve as critical effectors that promote infection upon entry into cells. How this dynamic network of protein interactions is arranged within virions is largely unknown. We present a molecular approach to dissect the tegument, and with it we begin to tease apart the protein interactions that underlie this complex layer of the virion architecture.

Pseudorabies virus induces autophagy to enhance viral replication in mouse neuro-2a cells in vitro.

Autophagy of cytoplasmic components plays an essential role in the pathogenic infection process. Furthermore, research suggests that autophagy is an extremely important component of the innate immune response. Our study aimed to reveal the effect of virus-induced autophagy on pseudorabies virus (PRV) replication. Our results confirmed that light chain 3 (LC3)-I was converted into LC3-II after PRV infection; this transition is considered an important indicator of autophagy. Transmission electron microscopy (TEM) revealed that PRV infection could notably increase the number of autophagosomes in mouse neuro-2a (N2a) cells. In addition, LC3-II accumulated in response to chloroquine (CQ) treatment, indicating that PRV infection induced a complete autophagic flux response. Furthermore, our analyses verified differences in the magnitude of autophagy induction by two different PRV isolates, LA and ZJ01. Subsequent analysis showed that the induction of autophagy by rapamycin facilitated PRV replication, while inhibition of autophagy by 3-methyladenine (3-MA) reduced PRV replication. These results indicated that PRV induced autophagy via the classical Beclin-1-Atg7-Atg5 pathway to enhance viral replication in N2a cells in vitro.

A pUL25 dimer interfaces the pseudorabies virus capsid and tegument.

Inside the virions of α-herpesviruses, tegument protein pUL25 anchors the tegument to capsid vertices through direct interactions with tegument proteins pUL17 and pUL36. In addition to promoting virion assembly, both pUL25 and pUL36 are critical for intracellular microtubule-dependent capsid transport. Despite these essential roles during infection, the stoichiometry and precise organization of pUL25 and pUL36 on the capsid surface remain controversial due to the insufficient resolution of existing reconstructions from cryo-electron microscopy (cryoEM). Here, we report a three-dimensional (3D) icosahedral reconstruction of pseudorabies virus (PRV), a varicellovirus of the α-herpesvirinae subfamily, obtained by electron-counting cryoEM at 4.9 Å resolution. Our reconstruction resolves a dimer of pUL25 forming a capsid-associated tegument complex with pUL36 and pUL17 through a coiled coil helix bundle, thus correcting previous misinterpretations. A comparison between reconstructions of PRV and the γ-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) reinforces their similar architectures and establishes important subfamily differences in the capsid-tegument interface.

Exocytosis of Alphaherpesvirus Virions, Light Particles, and Glycoproteins Uses Constitutive Secretory Mechanisms.

UNLABELLED: Many molecular and cell biological details of the alphaherpesvirus assembly and egress pathway remain unclear. Recently we developed a live-cell fluorescence microscopy assay of pseudorabies virus (PRV) exocytosis, based on total internal reflection fluorescence (TIRF) microscopy and a virus-encoded pH-sensitive fluorescent probe. Here, we use this assay to distinguish three classes of viral exocytosis in a nonpolarized cell type: (i) trafficking of viral glycoproteins to the plasma membrane, (ii) exocytosis of viral light particles, and (iii) exocytosis of virions. We find that viral glycoproteins traffic to the cell surface in association with constitutive secretory Rab GTPases and exhibit free diffusion into the plasma membrane after exocytosis. Similarly, both virions and light particles use these same constitutive secretory mechanisms for egress from infected cells. Furthermore, we show that viral light particles are distinct from cellular exosomes. Together, these observations shed light on viral glycoprotein trafficking steps that precede virus particle assembly and reinforce the idea that virions and light particles share a biogenesis and trafficking pathway. IMPORTANCE: The alphaherpesviruses, including the important human pathogens herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV), are among the few viruses that have evolved to exploit the mammalian nervous system. These viruses typically cause mild recurrent herpetic or zosteriform lesions but can also cause debilitating herpes encephalitis, more frequently in very young, old, immunocompromised, or nonnatural hosts. Importantly, many of the molecular and cellular mechanisms of viral assembly and egress remain unclear. This study addresses the trafficking of viral glycoproteins to the plasma membrane, exocytosis of light particles, and exocytosis of virions. Trafficking of glycoproteins affects immune evasion and pathogenesis and may precede virus particle assembly. The release of light particles may also contribute to immune evasion and pathogenesis. Finally, exocytosis of virions is important to understand, as this final step in the virus replication cycle produces infectious extracellular particles capable of spreading to the next round of host cells.

Extensive subunit contacts underpin herpesvirus capsid stability and interior-to-exterior allostery.

The herpesvirus capsid is a complex protein assembly that includes hundreds of copies of four major subunits and lesser numbers of several minor proteins, all of which are essential for infectivity. Cryo-electron microscopy is uniquely suited for studying interactions that govern the assembly and function of such large functional complexes. Here we report two high-quality capsid structures, from human herpes simplex virus type 1 (HSV-1) and the animal pseudorabies virus (PRV), imaged inside intact virions at ~7-Å resolution. From these, we developed a complete model of subunit and domain organization and identified extensive networks of subunit contacts that underpin capsid stability and form a pathway that may signal the completion of DNA packaging from the capsid interior to outer surface, thereby initiating nuclear egress. Differences in the folding and orientation of subunit domains between herpesvirus capsids suggest that common elements have been modified for specific functions.

Simultaneous Visualization of Parental and Progeny Viruses by a Capsid-Specific HaloTag Labeling Strategy.

Real-time, long-term, single-particle tracking (SPT) provides us an opportunity to explore the fate of individual viruses toward understanding the mechanisms underlying virus infection, which in turn could lead to the development of therapeutics against viral diseases. However, the research focusing on the virus assembly and egress by SPT remains a challenge because established labeling strategies could neither specifically label progeny viruses nor make them distinguishable from the parental viruses. Herein, we have established a temporally controllable capsid-specific HaloTag labeling strategy based on reverse genetic technology. VP26, the smallest pseudorabies virus (PrV) capsid protein, was fused with HaloTag protein and labeled with the HaloTag ligand during virus replication. The labeled replication-competent recombinant PrV harvested from medium can be applied directly in SPT experiments without further modification. Thus, virus infectivity, which is critical for the visualization and analysis of viral motion, is retained to the largest extent. Moreover, progeny viruses can be distinguished from parental viruses using diverse HaloTag ligands. Consequently, the entire course of virus infection and replication can be visualized continuously, including virus attachment and capsid entry, transportation of capsids to the nucleus along microtubules, docking of capsids on the nucleus, endonuclear assembly of progeny capsids, and the egress of progeny viruses. In combination with SPT, the established strategy represents a versatile means to reveal the mechanisms and dynamic global picture of the life cycle of a virus.

3D rotating wall vessel and 2D cell culture of four veterinary virus pathogens: A comparison of virus yields, portions of infectious particles and virus growth curves.

Only very few comparative studies have been performed that evaluate general trends of virus growth under 3D in comparison with 2D cell culture conditions. The aim of this study was to investigate differences when four animal viruses are cultured in 2D and 3D. Suid herpesvirus 1 (SuHV-1), Vesicular stomatitis virus (VSIV), Bovine adenovirus (BAdV) and Bovine parainfluenza 3 virus (BPIV-3) were cultivated in 3D rotating wall vessels (RWVs) and conventional 2D cultures. The production of virus particles, the portion of infectious particles, and the infectious growth curves were compared. For all viruses, the production of virus particles (related to cell density), including the non-infectious ones, was lower in 3D than in 2D culture. The production of only infectious particles was significantly lower in BAdV and BPIV-3 in 3D cultures in relation to cell density. The two cultivation approaches resulted in significantly different virus particle-to-TCID50 ratios in three of the four viruses: lower in SuHV-1 and BPIV-3 and higher in BAdV in 3D culture. The infectious virus growth rates were not significantly different in all viruses. Although 3D RWV culture resulted in lower production of virus particles compared to 2D systems, the portion of infectious particles was higher for some viruses.

Pseudorabies in farmed foxes fed pig offal in Shandong province, China.

Pseudorabies (PR, Aujeszky's disease) is an acute, highly contagious viral disease resulting in major economic losses to the swine industry. PR is endemic in wild and domestic animals, although its natural host is the pig. Here, we report an outbreak of PR in foxes on a fur-producing farm in Yuncheng county, Shandong, China, that were fed pig offal. The diagnosis of PR was based on nervous signs and standard PCR methods and by isolation of PRV from fox brain tissue in Vero cells. The diagnosis was confirmed by an indirect immunofluorescence assay and electron microscopy. Phylogenetic analysis of a partial (804 nt) viral glycoprotein gC gene sequence indicated that it was likely to be a field strain closely related to a cluster of PRV previously identified in China.

Antiviral Activity of Graphene Oxide: How Sharp Edged Structure and Charge Matter.

Graphene oxide and its derivatives have been widely explored for their antimicrobial properties due to their high surface-to-volume ratios and unique chemical and physical properties. However, little information is available on their effects on viruses. In this study, we report the broad-spectrum antiviral activity of GO against pseudorabies virus (PRV, a DNA virus) and porcine epidemic diarrhea virus (PEDV, an RNA virus). Our results showed that GO significantly suppressed the infection of PRV and PEDV for a 2 log reduction in virus titers at noncytotoxic concentrations. The potent antiviral activity of both GO and rGO can be attributed to the unique single-layer structure and negative charge. First, GO exhibited potent antiviral activity when conjugated with PVP, a nonionic polymer, but not when conjugated with PDDA, a cationic polymer. Additionally, the precursors Gt and GtO showed much weaker antiviral activity than monolayer GO and rGO, suggesting that the nanosheet structure is important for antiviral properties. Furthermore, GO inactivated both viruses by structural destruction prior to viral entry. The overall results suggest the potential of graphene oxide as a novel promising antiviral agent with a broad and potent antiviral activity.

Dimerization-Induced Allosteric Changes of the Oxyanion-Hole Loop Activate the Pseudorabies Virus Assemblin pUL26N, a Herpesvirus Serine Protease.

Herpesviruses encode a characteristic serine protease with a unique fold and an active site that comprises the unusual triad Ser-His-His. The protease is essential for viral replication and as such constitutes a promising drug target. In solution, a dynamic equilibrium exists between an inactive monomeric and an active dimeric form of the enzyme, which is believed to play a key regulatory role in the orchestration of proteolysis and capsid assembly. Currently available crystal structures of herpesvirus proteases correspond either to the dimeric state or to complexes with peptide mimetics that alter the dimerization interface. In contrast, the structure of the native monomeric state has remained elusive. Here, we present the three-dimensional structures of native monomeric, active dimeric, and diisopropyl fluorophosphate-inhibited dimeric protease derived from pseudorabies virus, an alphaherpesvirus of swine. These structures, solved by X-ray crystallography to respective resolutions of 2.05, 2.10 and 2.03 Å, allow a direct comparison of the main conformational states of the protease. In the dimeric form, a functional oxyanion hole is formed by a loop of 10 amino-acid residues encompassing two consecutive arginine residues (Arg136 and Arg137); both are strictly conserved throughout the herpesviruses. In the monomeric form, the top of the loop is shifted by approximately 11 Å, resulting in a complete disruption of the oxyanion hole and loss of activity. The dimerization-induced allosteric changes described here form the physical basis for the concentration-dependent activation of the protease, which is essential for proper virus replication. Small-angle X-ray scattering experiments confirmed a concentration-dependent equilibrium of monomeric and dimeric protease in solution.

Structure of the pseudorabies virus capsid: comparison with herpes simplex virus type 1 and differential binding of essential minor proteins.

The structure of pseudorabies virus (PRV) capsids isolated from the nucleus of infected cells and from PRV virions was determined by cryo-electron microscopy (cryo-EM) and compared to herpes simplex virus type 1 (HSV-1) capsids. PRV capsid structures closely resemble those of HSV-1, including distribution of the capsid vertex specific component (CVSC) of HSV-1, which is a heterodimer of the pUL17 and pUL25 proteins. Occupancy of CVSC on all PRV capsids is near 100%, compared to ~50% reported for HSV-1 C-capsids and 25% or less that we measure for HSV-1 A- and B-capsids. A PRV mutant lacking pUL25 does not produce C-capsids and lacks visible CVSC density in the cryo-EM-based reconstruction. A reconstruction of PRV capsids in which green fluorescent protein was fused within the N-terminus of pUL25 confirmed previous studies with a similar HSV-1 capsid mutant localizing pUL25 to the CVSC density region that is distal to the penton. However, comparison of the CVSC density in a 9-Å-resolution PRV C-capsid map with the available crystal structure of HSV-1 pUL25 failed to find a satisfactory fit, suggesting either a different fold for PRV pUL25 or a capsid-bound conformation for pUL25 that does not match the X-ray model determined from protein crystallized in solution. The PRV capsid imaged within virions closely resembles C-capsids with the addition of weak but significant density shrouding the pentons that we attribute to tegument proteins. Our results demonstrate significant structure conservation between the PRV and HSV capsids.

Glycoproteins gB and gH are required for syncytium formation but not for herpesvirus-induced nuclear envelope breakdown.

Herpesvirus nucleocapsids are assembled in the nucleus, whereas maturation into infectious virions takes place in the cytosol. Since, due to their size, nucleocapsids cannot pass the nuclear pores, they traverse the nuclear envelope by vesicle-mediated transport. Nucleocapsids bud at the inner nuclear membrane into the perinuclear space, forming primary enveloped particles and are released into the cytosol after fusion of the primary envelope with the outer nuclear membrane. The nuclear egress complex (NEC), consisting of the conserved herpesvirus proteins (p)UL31 and pUL34, is required for this process, whereas the viral glycoproteins gB and gH, which are essential for fusion during penetration, are not. We recently described herpesvirus-induced nuclear envelope breakdown (NEBD) as an alternative egress pathway used in the absence of the NEC. However, the molecular details of this pathway are still unknown. It has been speculated that glycoproteins involved in fusion during entry might play a role in NEBD. By deleting genes encoding glycoproteins gB and gH from the genome of NEBD-inducing pseudorabies viruses, we demonstrate that these glycoproteins are not required for NEBD but are still necessary for syncytium formation, again emphasizing fundamental differences in herpesvirus-induced alterations at the nuclear envelopes and plasma membranes of infected cells.

Attenuated live vaccine (Bartha-K16) caused pseudorabies (Aujeszky's disease) in sheep.

Pseudorabies (PR, Aujeszky's disease) is an acute, contagious viral disease that affects a wide range of domestic and wild species. In the present study, an outbreak of PR in sheep induced by attenuated live vaccine (Bartha-K16) was diagnosed and analyzed in China. The presence of PR virus (PRV) in brain samples from infected sheep was confirmed by gC gene polymerase chain reaction amplification and PRV-like particle observation by electron microscopy. The molecular characterization of the PRV was performed with homology analysis and phylogenetic tree construction based on the gC gene. This is the first description of an outbreak of PR induced by a live attenuated vaccine (Bartha-K16), indicating that this vaccine is unsuitable for use in sheep.

Differential protein partitioning within the herpesvirus tegument and envelope underlies a complex and variable virion architecture.

The herpesvirus virion is a multilayered structure consisting of a DNA-filled capsid, tegument, and envelope. Detailed reconstructions of the capsid are possible based on its icosahedral symmetry, but the surrounding tegument and envelope layers lack regular architecture. To circumvent limitations of symmetry-based ultrastructural reconstruction methods, a fluorescence approach was developed using single-particle imaging combined with displacement measurements at nanoscale resolution. An analysis of 11 tegument and envelope proteins defined the composition and plasticity of symmetric and asymmetric elements of the virion architecture. The resulting virion protein map ascribes molecular composition to density profiles previously acquired by traditional ultrastructural methods, and provides a way forward to examine the dynamics of the virion architecture during infection.

Pseudorabies virus and herpes simplex virus type 1 utilize different tegument-glycoprotein interactions to mediate the process of envelopment.

BACKGROUND AND OBJECTIVE: During herpesvirus envelopment capsids, tegument polypeptides and membrane proteins assemble at the site of budding, and a cellular lipid bilayer becomes refashioned into a spherical envelope. A web of interactions between tegument proteins and the cytoplasmic tails of viral glycoproteins play a critical role in this process. We have previously demonstrated that for herpes simplex virus (HSV)-1 the cytoplasmic tail of glycoprotein H (gH) binds the tegument protein VP16. The HSV and pseudorabies virus (PRV) genomes are essentially collinear, and individual gene products show significant sequence homology. However, the demarcation of function often differs between PRV and HSV proteins. The goal of this study was to determine whether PRV gH and VP16 interact in a manner similar to their homologs in HSV. METHODS: A fusion protein pull-down assay was performed in which a PRV gH cytoplasmic tail-glutathione S-transferase fusion protein, bound to glutathione-Sepharose beads, was incubated with PRV-infected cell cytosol, washed and subjected to Western blot analysis using anti-PRV VP16 antisera. RESULTS: Western blots indicate that PRV VP16 does not specifically bind to the PRV gH tail. CONCLUSION: Our results highlight that, despite the relatively close evolutionary relationship between HSV and PRV, there are significant differences in their protein interactions that drive envelopment.

Growth, physicochemical properties, and morphogenesis of Chinese wild-type PRV Fa and its gene-deleted mutant strain PRV SA215.

BACKGROUND: PRV Fa is common in China and causes most of the pseudorabies in the pig industry. A PRV SA215 strain with deleted gE, gI, and TK genes was constructed to develop a commercial attenuated live vaccine. However, the physicochemical properties, growth pattern, penetration kinetics, and morphogenesis of the PRV SA215 and its parental PRV Fa strain are unclear. RESULTS: A series of experiments were conducted to characterize both strains and provide more information. PRV Fa and PRV SA215 were found to have similar penetration patterns, with about 5 min half-time of penetration. The SA215 strain exhibited a slight delay in entry compared with PRV Fa. In the one-step growth test, the titers of the SA215 strain were first detected at 8 h, rapidly increased, and peaked at 12 h. A plateau was formed between 12-36 h of culturing. PRV SA215 showed delayed replication and approximately 10-30-fold lower titers during 0-16 h of culturing compared with the PRV-Fa strain. After 16 h, the PRV Fa titers dramatically decreased, whereas those of PRV SA215 were prolonged to 36 h and reached a titer value equal to that of PRV Fa and then decreased. Both strains were sensitive to both heat and acid-alkali treatments; however, PRV Fa was relatively more stable to heat treatment than PRV SA215. Both strains could propagate in the cultures with pH values from 5.0 to 9.0. Cultures with pH below 3.0 or above 11.0 were fatal to both strains. Both strains had considerable resistance to freeze-thawing treatments. Morphogenetic investigations showed that typical phases in the maturation pathway were observed in the PRV Fa-infected PK15 cells, whereas secondary envelopment was not observed in the PRV SA215 strain. Instead, capsid aggregations with concomitants of electrodense materials were observed. CONCLUSIONS: These results suggest that PRV SA215 is a promising strain for vaccine development.

Completely assembled virus particles detected by transmission electron microscopy in proximal and mid-axons of neurons infected with herpes simplex virus type 1, herpes simplex virus type 2 and pseudorabies virus.

The morphology of alphaherpesviruses during anterograde axonal transport from the neuron cell body towards the axon terminus is controversial. Reports suggest that transport of herpes simplex virus type 1 (HSV-1) nucleocapsids and envelope proteins occurs in separate compartments and that complete virions form at varicosities or axon termini (subassembly transport model), while transport of a related alphaherpesvirus, pseudorabies virus (PRV) occurs as enveloped capsids in vesicles (assembled transport model). Transmission electron microscopy of proximal and mid-axons of primary superior cervical ganglion (SCG) neurons was used to compare anterograde axonal transport of HSV-1, HSV-2 and PRV. SCG cell bodies were infected with HSV-1 NS and 17, HSV-2 2.12 and PRV Becker. Fully assembled virus particles were detected intracellularly within vesicles in proximal and mid-axons adjacent to microtubules after infection with each virus, indicating that assembled virions are transported anterograde within axons for all three alphaherpesviruses.

Random transposon-mediated mutagenesis of the essential large tegument protein pUL36 of pseudorabies virus.

Homologs of the pseudorabies virus (PrV) essential large tegument protein pUL36 are conserved throughout the Herpesviridae. pUL36 functions during transport of the nucleocapsid to and docking at the nuclear pore as well as during virion formation after nuclear egress in the cytoplasm. Deletion analyses revealed several nonessential regions within the 3,084-amino-acid PrV pUL36 (S. Böttcher, B. G. Klupp, H. Granzow, W. Fuchs, K. Michael, and T. C. Mettenleiter, J. Virol. 80:9910-9915, 2006; S. Böttcher, H. Granzow, C. Maresch, B. Möhl, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 81:13403-13411, 2007), while the C-terminal 62 amino acids are essential for virus replication (K. Coller, J. Lee, A. Ueda, and G. Smith, J. Virol. 81:11790-11797, 2007). To identify additional functional domains, we performed random mutagenesis of PrV pUL36 by transposon-mediated insertion of a 15-bp linker. By this approach, 26 pUL36 insertion mutants were selected and tested in transient transfection assays for their ability to complement one-step growth and/or viral spread of a PrV UL36 null mutant. Ten insertion mutants in the N-terminal half and 10 in the C terminus complemented both, whereas six insertion mutants clustering in the center of the protein did not complement in either assay. Interestingly, several insertions within conserved parts yielded positive complementation, including those located within the essential C-terminal 62 amino acids. For 15 mutants that mediated productive replication, stable virus recombinants were isolated and further characterized by plaque assay, in vitro growth analysis, and electron microscopy. Except for three mutant viruses, most insertion mutants replicated like wild-type PrV. Two insertion mutants, at amino acids (aa) 597 and 689, were impaired in one-step growth and viral spread and exhibited a defect in virion maturation in the cytoplasm. In contrast, one functional insertion (aa 1800) in a region which otherwise yielded only nonfunctional insertion mutants was impaired in viral spread but not in one-step growth without a distinctive ultrastructural phenotype. In summary, these studies extend and refine previous analyses of PrV pUL36 and demonstrate the different sensitivities of different regions of the protein to functional loss by insertion.