US3 Protein Research Articles

Exploitation of Herpesvirus Immune Evasion Strategies to Modify the Immunogenicity of Human Mesenchymal Stem Cell Transplants

BackgroundMesenchymal stem cells (MSCs) are multipotent cells residing in the connective tissue of many organs and holding great potential for tissue repair. In culture, human MSCs (hMSCs) are capable of extensive proliferation without showing chromosomal aberrations. Large numbers of hMSCs can thus be acquired from small samples of easily obtainable tissues like fat and bone marrow. MSCs can contribute to regeneration indirectly by secretion of cytokines or directly by differentiation into specialized cell types. The latter mechanism requires their long-term acceptance by the recipient. Although MSCs do not elicit immune responses in vitro, animal studies have revealed that allogeneic and xenogeneic MSCs are rejected.Methodology/Principal FindingsWe aim to overcome MSC immune rejection through permanent down-regulation of major histocompatibility complex (MHC) class I proteins on the surface of these MHC class II-negative cells through the use of viral immune evasion proteins. Transduction of hMSCs with a retroviral vector encoding the human cytomegalovirus US11 protein resulted in strong inhibition of MHC class I surface expression. When transplanted into immunocompetent mice, persistence of the US11-expressing and HLA-ABC-negative hMSCs at levels resembling those found in immunodeficient (i.e., NOD/SCID) mice could be attained provided that recipients' natural killer (NK) cells were depleted prior to cell transplantation.Conclusions/SignificanceOur findings demonstrate the potential utility of herpesviral immunoevasins to prevent rejection of xenogeneic MSCs. The observation that down-regulation of MHC class I surface expression renders hMSCs vulnerable to NK cell recognition and cytolysis implies that multiple viral immune evasion proteins are likely required to make hMSCs non-immunogenic and thereby universally transplantable.

Role of group A p21-activated kinases in the anti-apoptotic activity of the pseudorabies virus US3 protein kinase

The alphaherpesvirus US3 kinase is a conserved multifunctional serine/threonine kinase that plays a role in several processes, including modulation of the actin cytoskeleton, egress of virus particles from the nucleus and inhibition of apoptosis. However, the mechanisms used by the US3 protein to exert its functions remain poorly understood. Recently, we identified the group A p21-activated kinases PAK1 and PAK2 as important effectors in the US3-mediated cytoskeletal rearrangements. Here, we investigated if group A PAKs are also involved in the anti-apoptotic properties of US3. Infection experiments using a group A PAK inhibitor pointed at a moderate role for group A PAKs in the anti-apoptotic properties of US3. Furthermore, infection assays using wild type and US3null PRV in wild type MEF, PAK1 −/− MEF and PAK2 −/− MEF indicated that PAK2 does not play a role in US3-mediated inhibition of apoptosis during infection, whereas PAK1 plays a significant, yet limited role. Experiments in US3-transfected MEF using staurosporine as apoptosis trigger confirmed these observations. These results show that PAK1 plays a significant, yet limited, role in the anti-apoptotic activity of US3.

Keep it in the subfamily: the conserved alphaherpesvirus US3 protein kinase

The US3 protein kinase is conserved over the alphaherpesvirus subfamily. Increasing evidence shows that, although the kinase is generally not required for virus replication in cell culture, it plays a pivotal and in some cases an essential role in virus virulence in vivo. The US3 protein is a multifunctional serine/threonine kinase that is involved in viral gene expression, virion morphogenesis, remodelling the actin cytoskeleton and the evasion of several antiviral host responses. In the current review, both the well conserved and virus-specific functions of alphaherpesvirus US3 protein kinase orthologues will be discussed.

The p97 ATPase Dislocates MHC Class I Heavy Chain in US2-expressing Cells via a Ufd1-Npl4-independent Mechanism

The human cytomegalovirus (HCMV) protein US2 hijacks the endoplasmic reticulum (ER)-associated degradation machinery to dispose of MHC class I heavy chain (HC) at the ER. This process requires retrotranslocation of newly synthesized HC molecules from the ER membrane into the cytosol, but the mechanism underlying the dislocation reaction has been elusive. Here we establish an in vitro permeabilized cell assay that recapitulates the retrotranslocation of MHC HC in US2-expressing cells. Using this assay, we demonstrate that the dislocation process requires ATP and ubiquitin, as expected. The retrotranslocation also involves the p97 ATPase. However, the mechanism by which p97 dislocates MHC class I HC in US2 cells is distinct from that in US11 cells: the dislocation reaction in US2 cells is independent of the p97 cofactor Ufd1-Npl4. Our results suggest that different retrotranslocation mechanisms can employ distinct p97 ATPase complexes to dislocate substrates.

Circadian CLOCK histone acetyl transferase localizes at ND10 nuclear bodies and enables herpes simplex virus gene expression

Expression of herpes simplex virus genes at the initiation of replication involves two steps that take place at ND10 nuclear bodies. These are suppression of cellular repressors that attempt to silence viral DNA and remodeling of the viral chromatin to make it accessible for transcription. In earlier studies we reported on the mechanism by which viral proteins ICP0 and U(S)3 protein kinase modify and disrupt the HDAC1/CoREST/REST/LSD1 repressor complex. The remodeling step requires in addition acetylation of histones bound to DNA. In an attempt to identify the enzyme, we took note of the observation that ICP0 physically and functionally interacts with Bmal1, a partner of the CLOCK histone acetyl transferase, and key members of the bHLH-PAS family of transcriptional factors. The Bmal11 and CLOCK heterodimer is best known as a regulator of the circadian oscillation in the mammalian CLOCK system. In this article we report the following: (i) in infected cells both Bmal1 and CLOCK localize at ND10 bodies; (ii) wild-type virus stabilizes the CLOCK protein; (iii) overexpression of CLOCK partially compensates for the absence of ICP0 and enables higher yields in cells infected with a ΔICP0 mutant and this activity is not expressed by CLOCK mutants lacking histone acetyl transferase activity; and (iv) depletion of CLOCK in cells infected with wild-type virus results in significant decrease in the expression of all viral proteins tested. We conclude that ICP0 interacts with Bmal1 and by extension with CLOCK histone acetyl transferase to remodel viral chromatin.

Efficacy of an inactivated, recombinant bovine herpesvirus type 5 (BoHV-5) vaccine

Bovine herpesvirus type 5 (BoHV-5) is the causative agent of bovine herpetic encephalitis. In countries where BoHV-5 is prevalent, attempts to vaccinate cattle to prevent clinical signs from BoHV-5-induced disease have relied essentially on vaccination with BoHV-1 vaccines. However, such practice has been shown not to confer full protection to BoHV-5 challenge. In the present study, an inactivated, oil adjuvanted vaccine prepared with a recombinant BoHV-5 from which the genes coding for glycoprotein I ( gI), glycoprotein E ( gE) and membrane protein US9 were deleted (BoHV-5 gI/gE/US9 −), was evaluated in cattle in a vaccination/challenge experiment. The vaccine was prepared from a virus suspension containing a pre-inactivation antigenic mass equivalent to 10 7.69 TCID 50/dose. Three mL of the inactivated vaccine were administered subcutaneously to eight calves serologically negative for BoHV-5 (vaccinated group). Four other calves were mock-vaccinated with an equivalent preparation without viral antigens (control group). Both groups were boostered 28 days later. Neither clinical signs of disease nor adverse effects were observed during or after vaccination. A specific serological response, revealed by the development of neutralizing antibodies, was detected in all vaccinated animals after the first dose of vaccine, whereas control animals remained seronegative. Calves were subsequently challenged on day 77 post-vaccination (pv) with 10 9.25 TCID 50 of the wild-type BoHV-5 (parental strain EVI 88/95). After challenge, vaccinated cattle displayed mild signs of respiratory disease, whereas the control group developed respiratory disease and severe encephalitis, which led to culling of 2/4 calves. Searches for viral DNA in the central nervous system (CNS) of vaccinated calves indicated that wild-type BoHV-5 did not replicate, whereas in CNS tissues of calves on the control group, viral DNA was widely distributed. BoHV-5 shedding in nasal secretions was significantly lower in vaccinated calves than in the control group on days 2, 3, 4 and 6 post-challenge (pc). In addition, the duration of virus shedding was significantly shorter in the vaccinated (7 days) than in controls (12 days). Attempts to reactivate latent infection by administration of dexamethasone at 147 days pv led to recrudescence of mild signs of respiratory disease in both vaccinated and control groups. Infectious virus shedding in nasal secretions was detected at reactivation and was significantly lower in vaccinated cattle than in controls on days 11–13 post-reactivation (pr). It is concluded that the inactivated vaccine prepared with the BoHV-5 gI/gE/US9 − recombinant was capable of conferring protection to encephalitis when vaccinated cattle were challenged with a large infectious dose of the parental wild type BoHV-5. However, it did not avoid the establishment of latency nor impeded dexamethasone-induced reactivation of the virus, despite a significant reduction in virus shedding after challenge and at reactivation on vaccinated calves.

Molecular anatomy of subcellular localization of HSV-1 tegument protein US11 in living cells

The herpes simplex virus type I (HSV-1) US11 protein is an RNA-binding multifunctional regulator that specifically and stably associates with nucleoli. Although the C-terminal part of US11 was responsible for its nucleolar localization, the precise nucleolar localization signal (NoLS) and nuclear export signal (NES) of US11 and its nuclear import and export mechanisms are still elusive. In this study, fluorescence microscopy was employed to investigate the subcellular localization of US11 and characterize its transport mechanism in living cells. By constructing a series of deletion mutants fused with enhanced yellow fluorescent protein (EYFP), three novel NoLSs of US11 were for the first time mapped to amino acids 84–125, 126–152, and 89–146, respectively. Additionally, the NES was identified to locate between amino acids 89 and 119. Furthermore, the US11 protein was demonstrated to target to the cytoplasm through the NES by chromosomal region maintenance 1 (CRM1)-independent pathway, and to the nucleolus through Ran and importin β-dependent mechanism that does not require importin α5.

Pseudorabies Virus Tegument Protein Us2 Recruits the Mitogen-Activated Protein Kinase Extracellular-Regulated Kinase (ERK) to Membranes through Interaction with the ERK Common Docking Domain

The pseudorabies virus (PRV) Us2 protein binds to the extracellular-regulated kinase (ERK) and inhibits the activation of ERK nuclear targets by sequestering cytoplasmic ERK on cellular membranes. Utilizing a series of Us2 truncations, we determined that the minimal portion of Us2 required for interaction with ERK is contained within its amino-terminal 214 amino acids. The loss of the ability of Us2 to bind to ERK in coimmunoprecipitation experiments was accompanied by a failure of Us2 to form oligomers, raising the possibility that higher-order Us2 structures are required for ERK interaction. To map the Us2 interaction site on ERK, we introduced mutations into the region of ERK that interacts with the ERK kinase, MEK, or into the common docking (CD) domain that mediates interactions with many ERK substrates. ERK carrying mutations within the MEK binding region maintained the ability to bind Us2, whereas ERK carrying mutations within the CD domain did not. Furthermore, the ERK CD domain was required for the Us2-mediated recruitment of ERK to membranes. Taken together, these findings suggest that Us2 regulates ERK activity by spatially restricting ERK localization and also by interfering with select ERK-substrate interactions.

The nucleolus and viral infection

The nucleolus is a subnuclear structure of eukaryocytes. It was thought that nucleolus only participates in the biogenesis and processing of rRNA. However, more and more evidence shows that it has many other functions, such as tRNA precursor processing, stress sensing and it is also involved in gene silencing, senescence and cell cycle regulation. Here, we summarize the recent understandings about the nucleolar functions, the regulation of nucleolar localization of proteins and the role that the nucleolus plays in virus infection, in which some related studies of Herpes simplex virus type 1 (HSV-1) US11, UL24 and bovine herpesvirus-1 infected cell protein 27 (BICP27) carried out in our lab will also be included.

TRAM1 is involved in disposal of ER membrane degradation substrates

ER quality control consists of monitoring protein folding and targeting misfolded proteins for proteasomal degradation. ER stress results in an unfolded protein response (UPR) that selectively upregulates proteins involved in protein degradation, ER expansion, and protein folding. Given the efficiency in which misfolded proteins are degraded, there likely exist cellular factors that enhance the export of proteins across the ER membrane. We have reported that translocating chain-associated membrane protein 1 (TRAM1), an ER-resident membrane protein, participates in HCMV US2- and US11-mediated dislocation of MHC class I heavy chains (Oresic, K., Ng, C.L., and Tortorella, D. 2009). Consistent with the hypothesis that TRAM1 is involved in the disposal of misfolded ER proteins, cells lacking TRAM1 experienced a heightened UPR upon acute ER stress, as evidenced by increased activation of unfolded protein response elements (UPRE) and elevated levels of NF-κB activity. We have also extended the involvement of TRAM1 in the selective degradation of misfolded ER membrane proteins Cln6 M241T and US2, but not the soluble degradation substrate α 1-antitrypsin null HK. These degradation model systems support the paradigm that TRAM1 is a selective factor that can enhance the dislocation of ER membrane proteins.

Gene expression profiling of Pseudorabies virus (PrV) infected bovine cells by combination of transcript analysis and quantitative proteomic techniques

Infection of target cells by alphaherpesviruses leads to extensive modulation of host cell gene expression. To gain detailed information on the molecular pathways affected by infection of Madin–Darby bovine kidney (MDBK) cells with PrV, transcript analysis was combined with a stable isotope-based quantitative proteomic approach (SILAC). Four hours after infection cells were harvested and processed in parallel either for transcript analysis, for subcellular fractionation into nuclei and cytosol, for extraction of phosphoproteins, or for affinity extraction with Heparin Sepharose and Cibacron Blue F3G-A-Sepharose. All fractions were further analysed by large format two-dimensional gel electrophoresis in different pH-ranges to maximize the number of proteins to be identified and quantified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Cell fractionation was quick and easy to perform but in comparison to affinity fractionation yielded lower numbers of identified and quantified proteins. After infection with PrV, only two of the 55 proteins with significantly modulated protein levels showed significant changes in transcript levels, indicating that posttranslational modifications may play a major role in the cellular response to PrV infection. Application of isotope labelling to cell cultures infected with wild-type PrV-Ka and a US3 protein kinase negative mutant allowed to monitor pUS3-dependent changes in the expression levels of viral proteins pUL29, pUL39 and pUL42.

Pseudorabies virus US3-mediated inhibition of apoptosis does not affect infectious virus production

Preventing apoptosis during the early stages of infection of a host cell is generally thought to result in a higher yield of progeny virus. The US3 protein kinase of pseudorabies virus (PRV) and herpes simplex virus (HSV) is able to protect infected cells from apoptosis, which may be one of the reasons why both US3null PRV and US3null HSV replicate to lower virus titres in several cell types. However, such potential correlation between the higher amount of apoptosis in US3null virus-infected cells and the lower virus titres of US3null virus has not been investigated directly. In the current study, we found that a broad-spectrum caspase-inhibitor efficiently inhibited apoptosis in swine testicle and human laryngeal epidermoid carcinoma cells infected with US3null or wild-type (WT) PRV. However, inhibition of apoptosis did not affect US3null or WT PRV extracellular or cell-associated virus titres, nor did it restore the small plaque phenotype of US3null PRV.

Bovine herpesvirus-1 US3 protein kinase: critical residues and involvement in the phosphorylation of VP22

The US3 gene product of bovine herpesvirus-1 (BoHV-1) is a protein kinase that is expressed early during infection and capable of autophosphorylation. By examining differentially labelled US3 moieties by co-immunoprecipitation, we demonstrated that the protein kinase interacts with itself in vitro, which supports autophosphorylation by US3. Based on its homology to other serine/threonine protein kinases, we defined two highly conserved lysines in US3, at position 195 within the ATP-binding pocket and at position 282 within the catalytic loop; altering either residue resulted in kinase-dead mutants, demonstrating that these two residues are critical for the catalytic activity of BoHV-1 US3. During immunoprecipitation experiments, US3 interacted weakly with VP22, another tegument protein of BoHV-1. Furthermore, VP22 co-localized with US3 inside the nucleus in BoHV-1-infected cells. In vitro kinase assays demonstrated that VP22 is phosphorylated not only by US3, but also by the cellular casein kinase 2 (CK2) protein. The selective CK2 protein kinase inhibitor, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) and the less specific CK2 inhibitor Kenpaullone reduced VP22 phosphorylation, while CK1, protein kinase C or protein kinase A inhibitors did not affect phosphorylation. When US3 was included with VP22 in the kinase assay in the presence of DMAT, a low level of VP22 phosphorylation was observed. These data demonstrate that BoHV-1 VP22 interacts with both CK2 and US3, and that CK2 is the major kinase phosphorylating VP22, with US3 playing a minor role.

Effects of Phosphorylation of Herpes Simplex Virus 1 Envelope Glycoprotein B by Us3 Kinase In Vivo and In Vitro

We recently reported that the herpes simplex virus 1 (HSV-1) Us3 protein kinase phosphorylates threonine at position 887 (Thr-887) in the cytoplasmic tail of envelope glycoprotein B (gB) (A. Kato, J. Arii, I. Shiratori, H. Akashi, H. Arase, and Y. Kawaguchi, J. Virol. 83:250-261, 2009; T. Wisner, C. C. Wright, A. Kato, Y. Kawaguchi, F. Mou, J. D. Baines, R. J. Roller and D. C. Johnson, J. Virol. 83:3115-3126, 2009). In the studies reported here, we examined the effect(s) of this phosphorylation on viral replication and pathogenesis in vivo and present data showing that replacement of gB Thr-887 by alanine significantly reduced viral replication in the mouse cornea and development of herpes stroma keratitis and periocular skin disease in mice. The same effects have been reported for mice infected with a recombinant HSV-1 carrying a kinase-inactive mutant of Us3. These observations suggested that Us3 phosphorylation of gB Thr-887 played a critical role in viral replication in vivo and in HSV-1 pathogenesis. In addition, we generated a monoclonal antibody that specifically reacted with phosphorylated gB Thr-887 and used this antibody to show that Us3 phosphorylation of gB Thr-887 regulated subcellular localization of gB, particularly on the cell surface of infected cells.

Differences in the Regulatory and Functional Effects of the Us3 Protein Kinase Activities of Herpes Simplex Virus 1 and 2

Us3 protein kinases encoded by herpes simplex virus 1 (HSV-1) and 2 (HSV-2) are serine/threonine protein kinases and play critical roles in viral replication and pathogenicity in vivo. In the present study, we investigated differences in the biological properties of HSV-1 and HSV-2 Us3 protein kinases and demonstrated that HSV-2 Us3 did not have some of the HSV-1 Us3 kinase functions, including control of nuclear egress of nucleocapsids, localization of UL31 and UL34, and cell surface expression of viral envelope glycoprotein B. In agreement with the observations that HSV-2 Us3 was less important for these functions, the effect of HSV-2 Us3 kinase activity on virulence in mice following intracerebral inoculation was much lower than that of HSV-1 Us3. Furthermore, we showed that alanine substitution in HSV-2 Us3 at a site (aspartic acid at position 147) corresponding to one that can be autophosphorylated in HSV-1 Us3 abolished HSV-2 Us3 kinase activity. Thus, the regulatory and functional effects of Us3 kinase activity are different between HSV-1 and HSV-2.

Identification and Characterization of a Duck Enteritis Virus US3-Like Gene

Duck enteritis virus (DEV) causes substantial losses on duck farms; however, its molecular biology is poorly understood. Here, an open reading frame of a US3-like gene of DEV was identified from a DEV genomic library. Its existence was confirmed by cloning from DEV-infected duck embryo fibroblasts (DEFs) and DNA sequencing. The US3-like gene was then subcloned into a prokaryotic protein expression vector and expressed as a six-histidine-tagged fusion protein in Escherichia coli. The protein was purified and inoculated into rabbits for antiserum production. A primary antibody specific to the gene was successfully generated and used to detect the US3-like protein in DEV-infected duck cells. In vivo expression of the US3-like protein in DEV-infected DEFs was demonstrated with indirect immunofluorescence assay and regular fluorescence microscopy, whereas uninfected DEFs did not show any specific fluorescent staining. Furthermore, indirect immunofluorescence assay and confocal microscopy were used to study the time course and subcellular localization of the protein expression. The protein was found to be expressed as early as 2 hr postinfection, and its expression was increased by time at 4, 8, 12, and 24 hr postinfection. The protein was found to be localized mostly around the perinuclear area and in the cytosol, and also in the nucleus at later time points. In addition, a US3 protein phylogenetic tree was constructed and showed that the evolutionary relationship of DEV is close to the genus Mardivirus. In short, the DEV US3-like gene and its in vivo protein expression were found for the first time, and DEV classification and the gene's functions were suggested.

Major tegument protein VP8 of bovine herpesvirus 1 is phosphorylated by viral US3 and cellular CK2 protein kinases

The UL47 gene product, VP8, is one of the major tegument proteins of bovine herpesvirus 1 (BoHV-1) and is subject to phosphorylation. Analysis of protein bands co-immunoprecipitated with VP8 from BoHV-1-infected cells by mass spectroscopy suggested that VP8 interacts with two protein kinases: cellular CK2 and viral US3. CK2 is a highly conserved cellular protein, expressed ubiquitously and known to phosphorylate numerous proteins. The US3 gene product is one of the viral kinases produced by BoHV-1 during infection. Interactions of CK2 and US3 with VP8 were confirmed outside the context of infection when FLAG-VP8 was expressed alone or co-expressed with US3-haemagglutinin tag in Cos-7 cells. Furthermore, VP8 and US3 were found to co-localize in the nucleus during viral infection. To explore the significance of these interactions, an in vitro kinase assay was performed, which demonstrated that VP8 is heavily phosphorylated by CK2. In the presence of the highly specific CK2 kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), phosphorylation of VP8 was significantly reduced. Phosphorylation of VP8 was also inhibited by the presence of kenpaullone, a less specific CK2 inhibitor, but not by protein kinase CK1 or protein kinase C inhibitors. When VP8 and US3 were both included in the kinase assay in the presence of DMAT, phosphorylation of VP8 was again observed. Autophosphorylation of US3 was also detected and was not inhibited by DMAT. Based on these results, it is proposed that VP8 interacts with cellular CK2 and viral US3 in BoHV-1-infected cells, and is in turn subject to kinase activities associated with both of these proteins.

Investigation of the Mechanism by Which Herpes Simplex Virus Type 1 LAT Sequences Modulate Preferential Establishment of Latent Infection in Mouse Trigeminal Ganglia

We previously demonstrated that herpes simplex virus type 1 (HSV-1) preferentially establishes latent infection in monoclonal antibody (MAb) A5-positive ganglionic neurons and that a 2.8-kb portion of the HSV-1 genome, corresponding to the 5' end of the LAT (latency-associated transcript) coding region, is responsible for this phenotype (38, 65). In the current study we carried out further genetic mapping of this latency phenotype and investigated some of the mechanisms that might be responsible. Studies with the chimeric virus HSV-1 17syn+/LAT2, an HSV-1 virus engineered to express HSV-2 LAT, demonstrated that this virus exhibited an HSV-2 latency phenotype, preferentially establishing latency in MAb KH10-positive neurons. This result is complementary to that previously described for the chimeric virus HSV-2 333/LAT1 and indicate that the HSV-1 latency phenotype can be changed to that of HSV-2 by substitution of a 2.8-kb piece of complementary viral DNA. Sequential studies in which we evaluated the pattern of HSV-1 latent infection of the mouse trigeminal ganglion following ocular inoculation with viruses with deletions of functional thymidine kinase, glycoprotein E, ICP0, and US9 protein demonstrate that preferential establishment of HSV-1 latent infection in A5-positive neurons is not a consequence of (i) differential access of HSV-1 to A5-positive neurons,(ii) differential cell-to-cell spread of HSV-1 to A5-positive neurons, (iii) differential "round-trip" spread of HSV-1 to A5-positive neurons, or (iv) expression of ICP0. Additional mapping studies with the HSV-1 LAT deletion viruses dLAT371, 17DeltaSty, and 17Delta348 indicate that most of the LAT 5' exon is not required for HSV-1 to preferentially establish latent infection in A5-positive neurons.

Alphaherpesvirus US3-mediated reorganization of the actin cytoskeleton is mediated by group A p21-activated kinases

The US3 protein is a viral serine/threonine kinase that is conserved among all members of the Alphaherpesvirinae. The US3 protein of different alphaherpesviruses causes dramatic alterations in the actin cytoskeleton, such as the disassembly of actin stress fibers and formation of cell projections, which have been associated with increased intercellular virus spread. Here, we find that inhibiting group A p21-activated kinases (PAKs), which are key regulators in Cdc42/Rac1 Rho GTPase signaling pathways, impairs US3-mediated actin alterations. By using PAK1(-/-) and PAK2(-/-) mouse embryo fibroblasts (MEFs), we show that US3-mediated stress fiber disassembly requires PAK2, whereas US3-mediated cell projection formation mainly is mediated by PAK1, also indicating that PAK1 and PAK2 can have different biological effects on the organization of the actin cytoskeleton. In addition, US3 was found to bind and phosphorylate group A PAKs. Lack of group A PAKs in MEFs was correlated with inefficient virus spread. Thus, US3 induces its effect on the actin cytoskeleton via group A PAKs.

Comparison of the Pseudorabies Virus Us9 Protein with Homologs from Other Veterinary and Human Alphaherpesviruses

Pseudorabies virus (PRV) Us9 is a small, tail-anchored (TA) membrane protein that is essential for axonal sorting of viral structural proteins and is highly conserved among other members of the alphaherpesvirus subfamily. We cloned the Us9 homologs from two human pathogens, varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1), as well as two veterinary pathogens, equine herpesvirus type 1 (EHV-1) and bovine herpesvirus type 1 (BHV-1), and fused them to enhanced green fluorescent protein to examine their subcellular localization and membrane topology. Akin to PRV Us9, all of the Us9 homologs localized to the trans-Golgi network and had a type II membrane topology (typical of TA proteins). Furthermore, we examined whether any of the Us9 homologs could compensate for the loss of PRV Us9 in anterograde, neuron-to-cell spread of infection in a compartmented chamber system. EHV-1 and BHV-1 Us9 were able to fully compensate for the loss of PRV Us9, whereas VZV and HSV-1 Us9 proteins were unable to functionally replace PRV Us9 when they were expressed in a PRV background.