Nucleolin is required for an efficient herpes simplex virus type 1 infection

Productive infection by herpes simplex virus type 1 (HSV-1), which occurs in the host cell nucleus, is accompanied by dramatic modifications of the nuclear architecture, including profound alterations of nucleolar morphology. Here, we show that the three most abundant nucleolar proteins--nucleolin, B23, and fibrillarin--are redistributed out of the nucleoli as a consequence of HSV-1 infection. We show that the amount of nucleolin increases progressively during the course of infection. We demonstrate for the first time that a nucleolar protein, i.e., nucleolin, colocalizes with ICP8 in the viral replication compartments, at the time when viral replication is effective, suggesting an involvement of nucleolin in the HSV-1 DNA replication process. At later times of infection, a granular form of nucleolin localizes to the cytoplasm, in structures that display the characteristic features of aggresomes, indicating that this form of nucleolin is very probably destined for degradation. The delocalization of nucleolin from the nucleoli requires the viral ICP4 protein or a factor(s) whose expression involves ICP4. Using small interfering RNA technology, we show that viral replication requires a high level of nucleolin expression, demonstrating for the first time a direct role for a nucleolar protein in herpes simplex virus biology.     

Herpes simplex virus tegument protein US11 interacts with conventional kinesin heavy chain

Little is known about the mechanisms of transport of neurotropic herpesviruses, such as herpes simplex virus (HSV), varicella-zoster virus, and pseudorabies virus, within neurons. For these viruses, which replicate in the nucleus, anterograde transport from the cell body of dorsal root ganglion (DRG) neurons to the axon terminus occurs over long distances. In the case of HSV, unenveloped nucleocapsids in human DRG neurons cocultured with autologous skin were observed by immunoelectron microscopy to colocalize with conventional ubiquitous kinesin, a microtubule-dependent motor protein, in the cell body and axon during anterograde axonal transport. Subsequently, four candidate kinesin-binding structural HSV proteins were identified (VP5, VP16, VP22, and US11) using oligohistidine-tagged human ubiquitous kinesin heavy chain (uKHC) as bait. Of these viral proteins, a direct interaction between uKHC and US11 was identified. In vitro studies identified residues 867 to 894 as the US11-binding site in uKHC located within the proposed heptad repeat cargo-binding domain of uKHC. In addition, the uKHC-binding site in US11 maps to the C-terminal RNA-binding domain. US11 is consistently cotransported with kinetics similar to those of the capsid protein VP5 into the axons of dissociated rat neurons, unlike the other tegument proteins VP16 and VP22. These observations suggest a major role for the uKHC-US11 interaction in anterograde transport of unenveloped HSV nucleocapsids in axons.     

The US11 gene product of herpes simplex virus has intercellular trafficking activity.

The US11 gene product of herpes simplex virus is an abundant virion structural protein with RNA-binding regulatory activity. Its carboxyl-terminal half consists of tandem tripeptide repeats of the sequence RXP. We demonstrate that the US11 protein has intercellular trafficking activity and accumulates in the nucleolus when singly expressed in cultured cells, and that the RXP repeats are responsible for this activity. These same properties were also observed in cells expressing a fusion protein linking US11 to the green fluorescent protein. Furthermore, exogenous US11 protein was internalized by cells at 4 degrees C, which suggests that US11 protein uptake occurs primarily through an energy-independent pathway.     

The herpes simplex virus 1 RNA binding protein US11 is a virion component and associates with ribosomal 60S subunits.

The herpes simplex virus 1 US11 gene encodes a site- and conformation-specific RNA binding regulatory protein. We fused the coding sequence of this protein with that of beta-galactosidase, expressed the chimeric gene in Escherichia coli, and purified a fusion protein which binds RNA in the same way as the infected cell protein. The fusion protein was used to generate anti-US11 monoclonal antibody. Studies with this antibody showed that US11 protein is a viral structural protein estimated to be present in 600 to 1,000 copies per virion. The great majority of cytoplasmic US11 protein was found in association with the 60S subunit of infected cell ribosomes. US11 protein associates with ribosomes both late in infection at the time of its synthesis and at the time of infection after its introduction into the cytoplasm by the virion. US11 protein expressed in an uninfected cell line stably transfected with the US11 gene associates with ribosomal 60S subunits and localizes to nucleoli, suggesting that US11 protein requires no other viral functions for these associations.     

程序性细胞死亡因子4与单纯疱疹病毒US3蛋白相互作用及参与细胞凋亡

程序性细胞死亡因子-4（programmed celld eath-4,PDCD4）通过阻断相关基因的转录与翻译从而抑制肿瘤发生,单纯疱疹病毒-1（herpes simplex virus-1,HSV-1）US3蛋白激酶可有效调控病毒基因产物或外源因素引致的细胞凋亡。近期研究证明PDCD4在病毒感染细胞中以US3依赖及非依赖两种模式被磷酸化修饰,其中受US3修饰的PDCD4仍定位细胞核并随之被降解,这可能是细胞凋亡被抑制的主要原因之一,此外,PDCD4沉默可阻断复制不完全病毒引致的细胞凋亡,表明PDCD4与HSV-1 US3阻断细胞凋亡途径直接相关。本文综述了这两种蛋白及其作用关系的研究进展,为解析病毒与细胞相互作用机理提供新方向。     

The luminal domain of TGN38 interacts with integrin beta 1 and is involved in its trafficking

TGN38 luminal domain (TGN38LD) was expressed in Cos-7 cells to identify potential binding partners. The luminal domain was secreted but, surprisingly, a significant portion bound to the plasma membrane. Cells over-expressing TGN38LD or the full-length molecule detached from the substratum and left footprints positive for TGN38. Unexpectedly, in these cells, TGN38 colocalizes with integrin α5β1 at the Golgi, the cell surface or in the footprints and an increased amount of both integrin subunits on the plasma membrane was observed. Under physiological conditions when TGN38 is not overexpressed, it interacts with integrin β1. This was demonstrated by reciprocal co-immunoprecipitation of integrin β1 and TGN38. Functional analysis reveals that modification of the trafficking of TGN38 results in a parallel change in the distribution of integrin α5β1, leading to the conclusion that TGN38 is involved in the trafficking of integrin β1.     

A herpes simplex virus 1 US11-expressing cell line is resistant to herpes simplex virus infection at a step in viral entry mediated by glycoprotein D.

A baby hamster kidney [BHK(tk-)] cell line (US11cl19) which stably expresses the US11 and alpha 4 genes of herpes simplex virus 1 strain F [HSV-1(F)] was found to be resistant to infection with HSV-1. Although wild-type HSV-1(F) attached with normal kinetics to the surface of US11cl19 cells, most cells showed no evidence of infection and failed to accumulate detectable amounts of alpha mRNAs. The relationship between the expression of UL11 and resistance to HSV infection in US11cl19 cells has not been defined, but the block to infection with wild-type HSV-1 was overcome by exposing cells with attached virus on their surface to the fusogen polyethylene glycol, suggesting that the block to infection preceded the fusion of viral and cellular membranes. An escape mutant of HSV-1(F), designated R5000, that forms plaques on US11cl19 cells was selected. This mutant was found to contain a mutation in the glycoprotein D (gD) coding sequence that results in the substitution of the serine at position 140 in the mature protein to asparagine. A recombinant virus, designated R5001, was constructed in which the wild-type gD gene was replaced with the R5000 gD gene. The recombinant formed plaques on US11cl19 cells with an efficiency comparable to that of the escape mutant R5000, suggesting that the mutation in gD determines the ability of the mutant R5000 to grow on US11cl19 cells. The observation that the US11cl19 cells were slightly more resistant to fusion by polyethylene glycol than parental BHK(tk-) cells led to the selection and testing of clonal lines from unselected and polyethylene glycol-selected BHK(tk-) cells. The results were that 16% of unselected to as much as 36% of the clones selected for relative resistance to polyethylene glycol fusion exhibited various degrees of resistance to infection. The exact step at which the infection was blocked is not known, but the results illustrate the ease of selection of cell clones with one or more sites at which infection could be blocked.     

Heptad repeat 2 in herpes simplex virus 1 gH interacts with heptad repeat 1 and is critical for virus entry and fusion

Herpes simplex virus 1 (HSV-1) entry into cells and cell-cell fusion mediated by HSV-1 glycoproteins require four glycoproteins, gD, gB, gH, gL. Of these, gH is the only one that so far exhibits structural-functional features typical of viral fusion glycoproteins, i.e., a candidate fusion peptide and, downstream of it, a heptad repeat (HR) segment able to form a coiled coil, named HR-1. Here, we show that gH carries a functional HR-2 capable of physical interaction with HR-1. Specifically, mutational analysis of gH aimed at increasing or decreasing the ability of HR-2 to form a coiled coil resulted in an increase or decrease of fusion activity, respectively. HSV infection was modified accordingly. A mimetic peptide with the HR-2 sequence inhibited HSV-1 infection in a specific and dose-dependent manner. Circular dichroism spectroscopy showed that both HR-2 and HR-1 mimetic peptides adopt mainly random conformation in aqueous solution, while a decrease in peptide environmental polarity determines a conformational change, with a significant increase of the alpha-helical conformation content, in particular, for the HR-1 peptide. Furthermore, HR-1 and HR-2 mimetic peptides formed a stable complex, as revealed in nondenaturing electrophoresis and by circular dichroism. The mixture of HR-1 and HR-2 peptides reversed the inhibition of HSV infection exerted by the single peptides. Complex formation between HR-1 and HR-2 was independent of the presence of adjacent gH sequences and of additional glycoproteins involved in entry and fusion. Altogether, HR-2 adds to the features typical of class 1 fusion glycoproteins exhibited by HSV-1 gH.     

Packaging determinants in the UL11 tegument protein of herpes simplex virus type 1

The UL11 gene of herpes simplex virus type 1 encodes a 96-amino-acid tegument protein that is myristylated, palmitylated, and phosphorylated and is found on the cytoplasmic faces of nuclear, Golgi apparatus-derived, and plasma membranes of infected cells. Although this protein is thought to play a role in virus budding, its specific function is unknown. Purified virions were found to contain approximately 700 copies of the UL11 protein per particle, making it an abundant component of the tegument. Moreover, comparisons of cell-associated and virion-associated UL11 showed that packaging is selective for underphosphorylated forms, as has been reported for several other tegument proteins. Although the mechanism by which UL11 is packaged is unknown, previous studies have identified several sequence motifs in the protein that are important for membrane binding, intracellular trafficking, and interaction with UL16, another tegument protein. To ascertain whether any of these motifs are needed for packaging, a transfection/infection-based assay was used in which mutant forms of the protein must compete with the wild type. In this assay, the entire C-terminal half of UL11 was found to be dispensable. In the N-terminal half, the sites of myristylation and palmitylation, which enable membrane-binding and Golgi apparatus-specific targeting, were found to be essential for efficient packaging. The acidic cluster motif, which is not needed for Golgi apparatus-specific targeting but is involved in recycling the protein from the plasma membrane and for the interaction with UL16, was found to be essential, too. Thus, something other than mere localization of UL11 to Golgi apparatus-derived membranes is needed for packaging. The critical factor is unlikely to be the interaction with UL16 because other mutants that fail to bind this protein (due to removal of the dileucine-like motif or substitutions with foreign acidic clusters) were efficiently packaged. Collectively, these results suggest that UL11 packaging is not driven by a passive mechanism but instead requires trafficking through a specific pathway.     

Reconstitution of herpes simplex virus microtubule-dependent trafficking in vitro

Microtubule-mediated anterograde transport of herpes simplex virus (HSV) from the neuronal cell body to the axon terminal is crucial for the spread and transmission of the virus. It is therefore of central importance to identify the cellular and viral factors responsible for this trafficking event. In previous studies, we isolated HSV-containing cytoplasmic organelles from infected cells and showed that they represent the first and only destination for HSV capsids after they emerge from the nucleus. In the present study, we tested whether these cytoplasmic compartments were capable of microtubule-dependent traffic. Organelles containing green fluorescent protein-labeled HSV capsids were isolated and found to be able to bind rhodamine-labeled microtubules polymerized in vitro. Following the addition of ATP, the HSV-associated organelles trafficked along the microtubules, as visualized by time lapse microscopy in an imaging microchamber. The velocity and processivity of trafficking resembled those seen for neurotropic herpesvirus traffic in living axons. The use of motor-specific inhibitors indicated that traffic was predominantly kinesin mediated, consistent with the reconstitution of anterograde traffic. Immunocytochemical studies revealed that the majority of HSV-containing organelles attached to the microtubules contained the trans-Golgi network marker TGN46. This simple, minimal reconstitution of microtubule-mediated anterograde traffic should facilitate and complement molecular analysis of HSV egress in vivo.     

The herpes simplex virus type 1 (HSV-1) regulatory protein ICP0 interacts with and Ubiquitinates p53

Herpes simplex virus type 1 regulatory protein ICP0 contains a zinc-binding RING finger and has been shown to induce the proteasome-dependent degradation of a number of cellular proteins in a RING finger-dependent manner during infection. This domain of ICP0 is also required to induce the formation of unanchored polyubiquitin chains in vitro in the presence of ubiquitin-conjugating enzymes UbcH5a and UbcH6. These data indicate that ICP0 has the potential to act as a RING finger ubiquitin ubiquitin-protein isopeptide ligase (E3) and to induce the degradation of certain cellular proteins through ubiquitination and proteasome-mediated degradation. Here we demonstrate that ICP0 is a genuine RING finger ubiquitin E3 ligase that can interact with and mediate the ubiquitination of the major oncoprotein p53 both in vitro and in vivo. Ubiquitination of p53 requires ICP0 to have an intact RING finger domain and occurs independently of its ability to bind to the ubiquitin-specific protease USP7.     

Functional anatomy of herpes simplex virus 1 overlapping genes encoding infected-cell protein 22 and US1.5 protein

Earlier studies have shown that (i) the coding domain of the alpha22 gene encodes two proteins, the 420-amino-acid infected-cell protein 22 (ICP22) and a protein, US1.5, which is initiated from methionine 147 of ICP22 and which is colinear with the remaining portion of that protein; (ii) posttranslational processing of ICP22 mediated largely by the viral protein kinase UL13 yields several isoforms differing in electrophoretic mobility; and (iii) mutants lacking the carboxyl-terminal half of the ICP22 and therefore DeltaUS1.5 are avirulent and fail to express normal levels of subsets of both alpha (e.g., ICP0) or gamma2 (e.g., US11 and UL38) proteins. We have generated and analyzed two sets of recombinant viruses. The first lacked portions of or all of the sequences expressed solely by ICP22. The second set lacked 10 to 40 3'-terminal codons of ICP22 and US1. 5. The results were as follows. (i) In cells infected with mutants lacking amino-terminal sequences, translation initiation begins at methionine 147. The resulting protein cannot be differentiated in mobility from authentic US1.5, and its posttranslational processing is mediated by the UL13 protein kinase. (ii) Expression of US11 and UL38 genes by mutants carrying only the US1.5 gene is similar to that of wild-type parent virus. (iii) Mutants which express only US1. 5 protein are avirulent in mice. (iv) The coding sequences Met147 to Met171 are essential for posttranslational processing of the US1.5 protein. (v) ICP22 made by mutants lacking 15 or fewer of the 3'-terminal codons are posttranslationally processed whereas those lacking 18 or more codons are not processed. (vi) Wild-type and mutant ICP22 proteins localized in both nucleus and cytoplasm irrespective of posttranslational processing. We conclude that ICP22 encodes two sets of functions, one in the amino terminus unique to ICP22 and one shared by ICP22 and US1.5. These functions are required for viral replication in experimental animals. US1.5 protein must be posttranslationally modified by the UL13 protein kinase to enable expression of a subset of late genes exemplified by UL38 and US11. Posttranslational processing is determined by two sets of sequences, at the amino terminus and at the carboxyl terminus of US1.5, respectively, a finding consistent with the hypothesis that both domains interact with protein partners for specific functions.     

Binding partners for the UL11 tegument protein of herpes simplex virus type 1

The product of the U(L)11 gene of herpes simplex virus type 1 (HSV-1) is a 96-amino-acid tegument protein that accumulates on the cytoplasmic face of internal membranes. Although it is thought to be important for nucleocapsid envelopment and egress, the actual function of this protein is unknown. Previous studies focused on the characterization of sequence elements within the UL11 protein that function in membrane binding and trafficking to the Golgi apparatus. Binding was found to be mediated by two fatty acyl groups (myristate and palmitate), while an acidic cluster and a dileucine motif were identified as being important for the recycling of UL11 from the plasma membrane to the Golgi apparatus. The goal of the experiments described here was to identify and characterize binding partners (viral or cellular) of UL11. Using both immunoprecipitation and glutathione S-transferase (GST) pull-down assays, we identified a 40-kDa protein that specifically associates with UL11 from infected Vero cells. Mutational analyses revealed that the acidic cluster and the dileucine motif are required for this association, whereas the entire second half of UL11 is not. In addition, UL11 homologs from pseudorabies and Marek's disease herpesviruses were also found to be capable of binding to the 40-kDa protein from HSV-1-infected cells, suggesting that the interaction is conserved among alphaherpesviruses. Purification and analysis of the 40-kDa protein by mass spectrometry revealed that it is the product of the U(L)16 gene, a virion protein reported to be involved in nucleocapsid assembly. Cells transfected with a UL16-green fluorescent protein expression vector produced a protein that was of the expected size, could be pulled down with GST-UL11, and accumulated in a Golgi-like compartment only when coexpressed with UL11, indicating that the interaction does not require any other viral products. These data represent the first steps toward elucidating the network of tegument proteins that UL11 links to membranes.     

Binding of herpes simplex virus-1 US11 to specific RNA sequences

Herpes simplex virus-1 US11 is a RNA-binding protein with a novel RNA-binding domain. US11 has been reported to exhibit sequence- and conformation-specific RNA-binding, but the sequences and conformations important for binding are not known. US11 has also been described as a double-stranded RNA (dsRNA)-binding protein. To investigate the US11–RNA interaction, we performed in vitro selection of RNA aptamers that bind US11 from a RNA library consisting of >10¹⁴ 80 base sequences which differ in a 30 base randomized region. US11 bound specifically to selected aptamers with an affinity of 70 nM. Analysis of 23 selected sequences revealed a strong consensus sequence. The US11 RNA-binding domain and ≤46 bases of selected RNA containing the consensus sequence were each sufficient for binding. US11 binding protected the consensus motif from hydroxyl radical cleavage. RNase digestions of a selected aptamer revealed regions of both single-stranded RNA and dsRNA. We observed that US11 bound two different dsRNAs in a sequence non-specific manner, but with lower affinity than it bound selected aptamers. The results define a relatively short specific sequence that binds US11 with high affinity and indicate that dsRNA alone does not confer high-affinity binding.     

N-acetylgalactosaminyltransferase activity involved in O-glycosylation of herpes simplex virus type 1 glycoproteins

We report on N-acetylgalactosaminyltransferase (UDPacetylgalactosamine--protein acetylgalactosaminyltransferase; EC 2.4.1.41) activity in herpes simplex virus type 1 (HSV-1)-infected BHK and RicR14 cells, a line of ricin-resistant BHK cells defective in N-acetylglucosaminyltransferase I. The enzyme catalyzed the transfer of [14C]N-acetylgalactosamine (GalNAc) from UDP-[14C]GalNAc into HSV glycoproteins, as identified by immunoprecipitation. The sugar was selectively incorporated into the immature forms of herpesvirus glycoproteins pgC, pgD, and gA-pgB, which are known to contain N-linked glycans of the high-mannose type. The high incorporation of [14C]GalNAc into endogenous acceptors of HSV-1-infected RicR14 cells was consistent with the accumulation of immature forms of HSV glycoproteins which occurs in these cells. Mild alkaline borohydride treatment of glycoproteins labeled via GalNAc transferase showed that the transferred GalNAc was O-linked and represented the first sugar added to the peptide backbone.     

Cellular protein interactions with herpes simplex virus type 1 oriS.

The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains an AT-rich region and three highly homologous sequences, sites I, II, and III, identified as binding sites for the HSV-1 origin-binding protein (OBP). In the present study, interactions between specific oriS DNA sequences and proteins in uninfected cell extracts were characterized. The formation of one predominant protein-DNA complex, M, was demonstrated in gel shift assays following incubation of uninfected cell extracts with site I DNA. The cellular protein(s) that comprises complex M has been designated origin factor I (OF-I). The OF-I binding site was shown to partially overlap the OBP binding site within site I. Complexes with mobilities indistinguishable from that of complex M also formed with site II and III DNAs in gel shift assays. oriS-containing plasmid DNA mutated in the OF-I binding site exhibited reduced replication efficiency in transient assays, demonstrating a role for this site in oriS function. The OF-I binding site is highly homologous to binding sites for the cellular CCAAT DNA-binding proteins. The binding site for the CCAAT protein CP2 was found to compete for OF-I binding to site I DNA. These studies support a model involving the participation of cellular proteins in the initiation of HSV-1 DNA synthesis at oriS.     

Herpes simplex virus type 1 UL51 protein is involved in maturation and egress of virus particles

The UL51 gene of herpes simplex virus type 1 (HSV-1) encodes a phosphoprotein whose homologs are conserved throughout the herpes virus family. Recently, we reported that UL51 protein colocalizes with Golgi marker proteins in transfected cells and that targeting of UL51 protein to the Golgi apparatus depends on palmitoylation of its N-terminal cysteine at position 9 (N. Nozawa, T. Daikoku, T. Koshizuka, Y. Yamauchi, T. Yoshikawa, and Y. Nishiyama, J. Virol. 77:3204-3216, 2003). However, its role in the HSV replication cycle was unknown. Here, we generated UL51-null mutants (FDL51) in HSV-1 to uncover the function of UL51 protein. We show that the mutant plaques were much smaller in size and that maximal titers were reduced nearly 100-fold compared to wild-type virus. Electron microscopy indicated that the formation of nucleocapsids was not affected by the deletion of UL51 but that viral egress from the perinuclear space was severely compromised. In FDL51-infected cells, a large number of enveloped nucleocapsids were observed in the perinuclear space, but enveloped mature virions in the cytoplasm, as well as extracellular mature virions, were rarely detected. These defects were fully rescued by reinsertion of the UL51 gene. These results indicate that UL51 protein is involved in the maturation and egress of HSV-1 virus particles downstream of the initial envelopment step.     

14-3-3 protein interacts with Huntingtin-associated protein 1 and regulates its trafficking

HAP1 (Huntingtin-associated protein 1) consists of two alternately spliced isoforms (HAP1A and HAP1B, which have unique C-terminal sequences) and participates in intracellular trafficking. The C terminus of HAP1A is phosphorylated, and this phosphorylation was found to decrease the association of HAP1A with kinesin light chain, a protein involved in anterograde transport in cells. It remains unclear how this phosphorylation functions to regulate the association of HAP1 with trafficking proteins. Using the yeast two-hybrid system, we found that HAP1 also interacts with 14-3-3 proteins, which are involved in the assembly of protein complexes and the regulation of protein trafficking. The interaction of HAP1 with 14-3-3 is confirmed by their immunoprecipitation and colocalization in mouse brain. Moreover, this interaction is specific to HAP1A and is increased by the phosphorylation of the C terminus of HAP1A. We also found that expression of 14-3-3 decreases the association of HAP1A with kinesin light chain. As a result, there is less HAP1A distributed in neurite tips of PC12 cells that overexpress 14-3-3. Also, overexpression of 14-3-3 reduces the effect of HAP1A in promoting neurite outgrowth of PC12 cells. We propose that the phosphorylation-dependent interaction of HAP1A with 14-3-3 regulates HAP1 function by influencing its association with kinesin light chain and trafficking in neuronal processes.