Epstein-Barr virus signal transduction and B-lymphocyte growth transformation.

Latent EBV growth transformation of resting B-cells into indefinitely proliferating cell lines is a successful viral strategy for survival in its host and the basis of several human malignancies. EBV transforms cell growth through viral proteins that modify cell gene expression at the level of transcription or by appropriating signaling pathways. Analyses of the EBV-transforming protein LMP1 have begun to reveal that this receptor transduces critical signals by appropriating the TNF receptor signal transduction pathway to activate NF-kappaB and MAPK. While this has brought an important aspect into clearer focus, future progress in delineating the underlying mechanism of transformation, which will be essential to devising effective therapies to treat EBV-associated malignancies, will depend on resolving the intricacies of TRAF signal transduction. Since expression of cytokines, receptors, and anti-apoptotic proteins are regulated by TRAF signaling, another critical issue is delineating the genes that are specifically targeted by LMP1 in order to transform B-lymphocyte growth.

Identification of EBV transforming genes by recombinant EBV technology.

Epstein-Barr virus (EBV) is able to infect primary B-lymphocytes but usually does not proceed to replicate more virions. Instead, EBV persists as an incomplete virus and expresses 12 gene products that transform the growth of these cells into continuously proliferating lymphoblastoid cell lines. Because EBV is associated with several human malignancies, there is intense interest in delineating the molecular functions of these EBV gene products in transformation. This review focuses on the recombinant EBV technologies that have been developed to introduce specific mutations into EBV and test the functions of these EBV genes in primary B-lymphocyte growth transformation.

Epstein-Barr virus latent-infection membrane proteins are palmitoylated and raft-associated: protein 1 binds to the cytoskeleton through TNF receptor cytoplasmic factors.

Epstein-Barr virus encodes integral membrane proteins LMP1 and LMP2A in transformed lymphoblastoid cell lines. We now find that LMP1 associates with the cell cytoskeleton through a tumor necrosis factor receptor-associated factor-interacting domain, most likely mediated by tumor necrosis factor receptor-associated factor 3. LMP1 is palmitoylated, and the transmembrane domains associate with lipid rafts. Mutation of LMP1 cysteine-78 abrogates palmitoylation but does not affect raft association or NF-kappaB or c-Jun N-terminal kinase activation. LMP2A also associates with rafts and is palmitoylated but does not associate with the cell cytoskeleton. The associations of LMP1 and LMP2A with rafts and of LMP1 with the cell cytoskeleton are likely to effect interactions with cell proteins involved in shape, motility, signal transduction, growth, and survival.

Epstein-barr virus transformation: involvement of latent membrane protein 1-mediated activation of NF-kappaB.

Epstein-Barr virus (EBV) transforms resting primary human B lymphocytes into indefinitely proliferating lymphoblastoid cell lines in vitro and is associated with several human malignancies in vivo. Recombinant EBV genetic analyses combined with in vitro B lymphocyte transformation assays demonstrate that latent infection membrane protein 1 (LMP1) is essential for EBV-mediated lymphocyte transformation. LMP1 has no intrinsic enzymatic activity but instead aggregates cellular proteins of the tumor necrosis factor receptor signaling pathway to activate transcription factor NF-kappaB. Mutants rendering LMP1 defective in these protein interactions are impaired in their abilities to activate NF-kappaB in reporter gene assays. Concordantly, EBV recombinants with LMP1 mutations that are compromised for NF-kappaB activation are impaired for growth transformation. Thus, EBV-mediated growth transformation is genetically and biochemically linked to LMP1-mediated activation of NF-kappaB.

The residues between the two transformation effector sites of Epstein-Barr virus latent membrane protein 1 are not critical for B-lymphocyte growth transformation.

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for EBV-mediated transformation of primary B lymphocytes. LMP1 spontaneously aggregates in the plasma membrane and enables two transformation effector sites (TES1 and TES2) within the 200-amino-acid cytoplasmic carboxyl terminus to constitutively engage the tumor necrosis factor receptor (TNFR)-associated factors TRAF1, TRAF2, TRAF3, and TRAF5 and the TNFR-associated death domain proteins TRADD and RIP, thereby activating NF-kappaB and c-Jun N-terminal kinase (JNK). To investigate the importance of the 60% of the LMP1 carboxyl terminus that lies between the TES1-TRAF and TES2-TRADD and -RIP binding sites, an EBV recombinant was made that contains a specific deletion of LMP1 codons 232 to 351. Surprisingly, the deletion mutant was similar to wild-type (wt) LMP1 EBV recombinants in its efficiency in transforming primary B lymphocytes into lymphoblastoid cell lines (LCLs). Mutant and wt EBV-transformed LCLs were similarly efficient in long-term outgrowth and in regrowth after endpoint dilution. Mutant and wt LMP1 proteins were also similar in their constitutive association with TRAF1, TRAF2, TRAF3, TRADD, and RIP. Mutant and wt EBV-transformed LCLs were similar in steady-state levels of Bcl2, JNK, and activated JNK proteins. The wt phenotype of recombinants with LMP1 codons 232 to 351 deleted further demarcates TES1 and TES2, underscores their central importance in B-lymphocyte growth transformation, and provides a new perspective on LMP1 sequence variation between TES1 and TES2.

An Epstein-Barr virus that expresses only the first 231 LMP1 amino acids efficiently initiates primary B-lymphocyte growth transformation.

An Epstein-Barr virus (EBV) recombinant (MS231) that expresses the first 231 amino acids (aa) of LMP1 and is truncated 155 aa before the carboxyl terminus transformed resting B lymphocytes into lymphoblastoid cell lines (LCLs) only when the infected cells were grown on fibroblast feeder cells (K. M. Kaye et al., J. Virol. 69:675-683, 1995). Higher-titer MS231 virus has now been compared to wild-type (WT) EBV recombinants for the ability to cause resting primary B-lymphocyte transformation. Unexpectedly, MS231 is as potent as WT EBV recombinants in causing infected B lymphocytes to proliferate in culture for up to 5 weeks. When more than one transforming event is initiated in a microwell, the MS231 recombinant supports efficient long-term LCL outgrowth and fibroblast feeder cells are not required. However, with limited virus input, MS231-infected cells differed in their growth from WT virus-infected cells as early as 6 weeks after infection. In contrast to WT virus-infected cells, most MS231-infected cells could not be grown into long-term LCLs. Thus, the LMP1 amino-terminal 231 aa are sufficient for initial growth transformation but the carboxyl-terminal 155 aa are necessary for efficient long-term outgrowth. Despite the absence of the carboxyl-terminal 155 aa, MS231- and WT-transformed LCLs are similar in latent EBV gene expression, in ICAM-1 and CD23 expression, and in NF-kappaB and c-jun N-terminal kinase activation. MS231 recombinant-infected LCLs, however, require 16- to 64-fold higher cell density than WT-infected LCLs for regrowth after limiting dilution. These data indicate that the LMP1 carboxyl-terminal 155 aa are important for growth at lower cell density and appear to reduce dependence on paracrine growth factors.

The Epstein-Barr virus oncoprotein latent membrane protein 1 engages the tumor necrosis factor receptor-associated proteins TRADD and receptor-interacting protein (RIP) but does not induce apoptosis or require RIP for NF-kappaB activation.

A site in the Epstein-Barr virus (EBV) transforming protein LMP1 that constitutively associates with the tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein TRADD to mediate NF-kappaB and c-Jun N-terminal kinase activation is critical for long-term lymphoblastoid cell proliferation. We now find that LMP1 signaling through TRADD differs from TNFR1 signaling through TRADD. LMP1 needs only 11 amino acids to activate NF-kappaB or synergize with TRADD in NF-kappaB activation, while TNFR1 requires approximately 70 residues. Further, LMP1 does not require TRADD residues 294 to 312 for NF-kappaB activation, while TNFR1 requires TRADD residues 296 to 302. LMP1 is partially blocked for NF-kappaB activation by a TRADD mutant consisting of residues 122 to 293. Unlike TNFR1, LMP1 can interact directly with receptor-interacting protein (RIP) and stably associates with RIP in EBV-transformed lymphoblastoid cell lines. Surprisingly, LMP1 does not require RIP for NF-kappaB activation. Despite constitutive association with TRADD or RIP, LMP1 does not induce apoptosis in EBV-negative Burkitt lymphoma or human embryonic kidney 293 cells. These results add a different perspective to the molecular interactions through which LMP1, TRADD, and RIP participate in B-lymphocyte activation and growth.

Role of the TRAF binding site and NF-kappaB activation in Epstein-Barr virus latent membrane protein 1-induced cell gene expression.

In this study, we investigated the induction of cellular gene expression by the Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1). Previously, LMP1 was shown to induce the expression of ICAM-1, LFA-3, CD40, and EBI3 in EBV-negative Burkitt lymphoma (BL) cells and of the epidermal growth factor receptor (EGF-R) in epithelial cells. We now show that LMP1 expression also increased Fas and tumor necrosis factor receptor-associated factor 1 (TRAF1) in BL cells. LMP1 mediates NF-kappaB activation via two independent domains located in its C-terminal cytoplasmic tail, a TRAF-interacting site that associates with TRAF1, -2, -3, and -5 through a PXQXT/S core motif and a TRADD-interacting site. In EBV-transformed B cells or transiently transfected BL cells, significant amounts of TRAF1, -2, -3, and -5 are associated with LMP1. In epithelial cells, very little TRAF1 is expressed, and only TRAF2, -3, and -5, are significantly complexed with LMP1. The importance of TRAF binding to the PXQXT/S motif in LMP1-mediated gene induction was studied by using an LMP1 mutant that contains alanine point mutations in this motif and fails to associate with TRAFs. This mutant, LMP1(P204A/Q206A), induced 60% of wild-type LMP1 NF-kappaB activation and had approximately 60% of wild-type LMP1 effect on Fas, ICAM-1, CD40, and LFA-3 induction. In contrast, LMP1(P204A/Q206A) was substantially more impaired in TRAF1, EBI3, and EGF-R induction. Thus, TRAF binding to the PXQXT/S motif has a nonessential role in up-regulating Fas, ICAM-1, CD40, and LFA-3 expression and a critical role in up-regulating TRAF1, EBI3, and EGF-R expression. Further, D1 LMP1, an LMP1 mutant that does not aggregate failed to induce TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 expression confirming the essential role for aggregation in LMP1 signaling. Overexpression of a dominant form of IkappaBalpha blocked LMP1-mediated TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 up-regulation, indicating that NF-kappaB is an important component of LMP1-mediated gene induction from both the TRAF- and TRADD-interacting sites.

The Epstein-Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-kappaB.

The Epstein-Barr virus latent membrane protein 1 (LMP1) is essential for the transformation of B lymphocytes into lymphoblastoid cell lines. Previous data are consistent with a model that LMP1 is a constitutively activated receptor that transduces signals for transformation through its carboxyl-terminal cytoplasmic tail. One transformation effector site (TES1), located within the membrane proximal 45 residues of the cytoplasmic tail, constitutively engages tumor necrosis factor receptor-associated factors. Signals from TES1 are sufficient to drive initial proliferation of infected resting B lymphocytes, but most lymphoblastoid cells infected with a virus that does not express the 155 residues beyond TES1 fail to grow as long-term cell lines. We now find that mutating two tyrosines to an isoleucine at the carboxyl end of the cytoplasmic tail cripples the ability of EBV to cause lymphoblastoid cell outgrowth, thereby marking a second transformation effector site, TES2. A yeast two-hybrid screen identified TES2 interacting proteins, including the tumor necrosis factor receptor-associated death domain protein (TRADD). TRADD was the only protein that interacted with wild-type TES2 and not with isoleucine-mutated TES2. TRADD associated with wild-type LMP1 but not with isoleucine-mutated LMP1 in mammalian cells, and TRADD constitutively associated with LMP1 in EBV-transformed cells. In transfection assays, TRADD and TES2 synergistically mediated high-level NF-kappaB activation. These results indicate that LMP1 appropriates TRADD to enable efficient long-term lymphoblastoid cell outgrowth. High-level NF-kappaB activation also appears to be a critical component of long-term outgrowth.

The Epstein-Barr virus LMP1 amino acid sequence that engages tumor necrosis factor receptor associated factors is critical for primary B lymphocyte growth transformation.

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for transforming primary B lymphocytes into lymphoblastoid cell lines. EBV recombinants with LMP1 genes truncated after the proximal 45 codons of the LMP1 carboxyl terminus are adequate for transformation. The proximal 45 residues include a domain that engages the tumor necrosis factor receptor associated factors (TRAFs). We investigated the importance of the TRAF binding domain by assaying the transforming ability of recombinant EBV genomes with a deletion of LMP1 codons 185-211. This mutation eliminates TRAF association in yeast and in lymphoblasts but does not affect LMP1 stability or localization. Specifically mutated recombinant EBV genomes were generated by transfecting P3HR-1 cells with overlapping EBV cosmids. Infection of primary B lymphocytes resulted in cell lines that were coinfected with an LMP1 delta185-211 EBV recombinant and P3HR-1 EBV, which has a wild-type LMP1 gene but is transformation defective due to another deletion. Despite the equimolar mixture of wild-type and mutated LMP1 genes in virus preparations from five coinfected cell lines, only the wild-type LMP1 gene was found in 412 cell lines obtained after transformation of primary B lymphocytes. No transformed cell line had only the LMP1 delta185-211 gene. An EBV recombinant with a Flag-tagged LMP1 gene passaged in parallel segregated from the coinfecting P3HR-1. These data indicate that the LMP1 TRAF binding domain is critical for primary B lymphocyte growth transformation.

In vivo growth of Epstein-Barr virus transformed B cells with mutations in latent membrane protein 2 (LMP2).

Epstein-Barr virus (EBV) causes infectious mononucleosis in adolescents and is associated with malignant B lymphocyte proliferation in AIDS patients, patients undergoing immune suppression for organ transplantation, and SCID mice. In vitro, EBV transformed, latently infected lymphoblastoid B cell lines (LCLs) contain EBV episomes and express nine virus encoded proteins. Six are nuclear proteins (EBNAs) and three are the integral membrane proteins, LMP1, LMP2A, and LMP2B. To determine if LMP2 was essential for in vivo growth, SCID mice were injected with LCLs containing wild-type EBV (LMP2+) or with LCLs transformed with EBV containing mutations in either LMP2A or LMP2B (LMP2-). SCID mice injected with the LMP2+ or LMP2- LCLs were monitored for tumor development, length of time to tumor development, and phenotypic characterization of the resulting tumors. No difference was observed in any of the above parameters between LMP2+ and LMP2- LCLs demonstrating that LMP2 is not essential for the in vivo growth of EBV transformed B lymphocytes in SCID mice.

Association of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr virus LMP1 domain important for B-lymphocyte transformation: role in NF-kappaB activation.

The Epstein-Barr virus (EBV) transforming protein LMP1 appears to be a constitutively activated tumor necrosis factor receptor (TNFR) on the basis of an intrinsic ability to aggregate in the plasma membrane and an association of its cytoplasmic carboxyl terminus (CT) with TNFR-associated factors (TRAFs). We now show that in EBV-transformed B lymphocytes most of TRAF1 or TRAF3 and 5% of TRAF2 are associated with LMP1 and that most of LMP1 is associated with TRAF1 or TRAF3. TRAF1, TRAF2, and TRAF3 bind to a single site in the LMP1 CT corresponding to amino acids (aa) 199 to 214, within a domain which is important for B-lymphocyte growth transformation (aa 187 to 231). Further deletional and alanine mutagenesis analyses and comparison with TRAF binding sequences in CD40, in CD30, and in the LMP1 of other lymphycryptoviruses provide the first evidence that PXQXT/S is a core TRAF binding motif. The negative effects of point mutations in the LMP1(1-231) core TRAF binding motif on TRAF binding and NF-kappaB activation genetically link the TRAFs to LMP1(1-231)-mediated NF-kappaB activation. NF-kappaB activation by LMP1(1-231) is likely to be mediated by TRAF1/TRAF2 heteroaggregates since TRAF1 is unique among the TRAFs in coactivating NF-kappaB with LMP1(1-231), a TRAF2 dominant-negative mutant can block LMP1(1-231)-mediated NF-kappaB activation as well as TRAF1 coactivation, and 30% of TRAF2 is associated with TRAF1 in EBV-transformed B cells. TRAF3 is a negative modulator of LMP1(1-231)-mediated NF-kappaB activation. Surprisingly, TRAF1, -2, or -3 does not interact with the terminal LMP1 CT aa 333 to 386 which can independently mediate NF-kappaB activation. The constitutive association of TRAFs with LMP1 through the aa 187 to 231 domain which is important in NF-kappaB activation and primary B-lymphocyte growth transformation implicates TRAF aggregation in LMP1 signaling.

Tumor necrosis factor receptor associated factor 2 is a mediator of NF-kappa B activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein.

Latent infection membrane protein 1 (LMP1), the Epstein-Barr virus transforming protein, associates with tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) and TRAF3. Since TRAF2 has been implicated in TNFR-mediated NF-kappa B activation, we have evaluated the role of TRAF2 in LMP1-mediated NF-kappa B activation. TRAF2 binds in vitro to the LMP1 carboxyl-terminal cytoplasmic domain (CT), coprecipitates with LMP1 in B lymphoblasts, and relocalizes to LMP1 plasma membrane patches. A dominant negative TRAF2 deletion mutant that lacks amino acids 6-86 (TRAF/ delta 6-86) inhibits NF-kappa B activation from the LMP1 CT and competes with TRAF2 for LMP1 binding. TRAF2 delta 6-86 inhibits NF-kappa B activation mediated by the first 45 amino acids of the LMP1 CT by more than 75% but inhibits NF-kappa B activation through the last 55 amino acids of the CT by less than 40%. A TRAF interacting protein, TANK, inhibits NF-kappa B activation by more than 70% from both LMP1 CT domains. These data implicate TRAF2 aggregation in NF-kappa B activation by the first 45 amino acids of the LMP1 CT and suggest that a different TRAF-related pathway may be involved in NF-kappa B activation by the last 55 amino acids of the LMP1 CT.

The Epstein-Barr virus LMP1 cytoplasmic carboxy terminus is essential for B-lymphocyte transformation; fibroblast cocultivation complements a critical function within the terminal 155 residues.

Recombinant Epstein-Barr viruses (EBVs) were made with mutated latent membrane protein 1 (LMP1) genes that express only the LMP1 amino-terminal cytoplasmic and six transmembrane domains (MS187) or these domains and the first 44 amino acids of the 200-residue LMP1 carboxy-terminal domain (MS231). After infection of primary B lymphocytes with virus stocks having small numbers of recombinant virus and large numbers of P3HR-1 EBV which is transformation defective but wild type (WT) for LMP1, all lymphoblastoid cell lines (LCLs) that had MS187 or MS231 LMP1 also had WT LMP1 provided by the coinfecting P3HR-1 EBV. Lytic virus infection was induced in these coinfected LCLs, and primary B lymphocytes were infected. In over 200 second-generation LCLs, MS187 LMP1 was never present without WT LMP1. Screening of over 600 LCLs infected with virus from MS231 recombinant virus-infected LCLs identified two LCLs which were infected with an MS231 recombinant without WT LMP1. The MS231 recombinant virus could growth transform primary B lymphocytes when cells were grown on fibroblast feeders. Even after 6 months on fibroblast feeder layers, cells transformed by the MS231 recombinant virus died when transferred to medium without fibroblast feeder cells. These data indicate that the LMP1 carboxy terminus is essential for WT growth-transforming activity. The first 44 amino acids of the carboxy-terminal cytoplasmic domain probably include an essential effector of cell growth transformation, while a deletion of the rest of LMP1 can be complemented by growth on fibroblast feeder layers. LMP1 residues 232 to 386 therefore provide a growth factor-like effect for the transformation of B lymphocytes. This effect may be indicative of the broader role of LMP1 in cell growth transformation.

Epstein-Barr virus recombinant molecular genetic analysis of the LMP1 amino-terminal cytoplasmic domain reveals a probable structural role, with no component essential for primary B-lymphocyte growth transformation.

Previous recombinant Epstein-Barr virus molecular genetic experiments with specifically mutated LMP1 genes indicate that LMP1 is essential for primary B-lymphocyte growth transformation and that the amino-terminal cytoplasmic and first transmembrane domains are together an important mediator of transformation. EBV recombinants with specific deletions in the amino-terminal cytoplasmic domain have now been constructed and tested for the ability to growth transform primary B lymphocytes into lymphoblastoid cell lines. Surprisingly, deletion of DNA encoding EHDLER or GPPLSSS from the full LMP1 amino-terminal cytoplasmic domain (MEHDLERGPPGPRRPPRGPPLSSS) had no discernible effect on primary B-lymphocyte transformation. These two motifs distinguish the LMP1 amino-terminal cytoplasmic domain from other arginine-rich membrane proximal sequences that anchor hydrophobic transmembrane domains. Two deletions which included the ERGPPGPRRPPR motif adversely affected but did not prevent transformation. This arginine- and proline-rich sequence is probably important in anchoring the first transmembrane domain in the plasma membrane, since these mutated LMP1s had altered stability and cell membrane localization. The finding that overlapping deletions of the entire amino-terminal cytoplasmic domain do not ablate transformation is most consistent with a model postulating that the transmembrane and carboxyl-terminal cytoplasmic domains are the likely biochemical effectors of transformation.

Epstein-Barr virus latent membrane protein 1 is essential for B-lymphocyte growth transformation.

The gene encoding latent-infection membrane protein 1 (LMP1) was specifically mutated in Epstein-Barr virus (EBV) recombinants by inserting a nonsense linker after codon 9 or codon 84 or into an intron 186 bp 3' to the latter insertion site. EBV recombinants with the LMP1 intron mutation were wild type for LMP1 expression and for growth transformation of primary B lymphocytes. In contrast, EBV recombinants with the mutations in the LMP1 open reading frame expressed N-terminally truncated crossreactive proteins and could initiate or maintain primary B-lymphocyte transformation only when wild-type LMP1 was provided in trans by a coinfecting, transformation-defective EBV, P3HR-1. These data indicate that LMP1 is essential for EBV-mediated transformation of primary B lymphocytes, that the first 43 amino acids are critical for LMP1's function, and that codon 44-initiated LMP1 does not have a dominant negative effect on transformation.

Delineation of the cis-acting element mediating EBNA-2 transactivation of latent infection membrane protein expression.

To delineate the cis-acting element through which EBNA-2 transactivates latent membrane protein 1 (LMP1), we assayed the effect of EBNA-2 on the activity of LMP1 promoter upstream deletion mutants in the context of the LMP1 or heterologous promoters controlling chloramphenicol acetyltransferase (CAT) reporter gene expression in Epstein-Barr virus-negative Burkitt lymphoma cells. Assays of progressive 5' deletions of the LMP1 promoter revealed low constitutive and at least eightfold EBNA-2-stimulated activity from -512 to +40 (-512/+40), -334/+40, and -234/+40 LMP1CAT plasmids. More extensive 5'-deleted -205/+40, -155/+40, and -147/+40 LMP1CAT plasmids also had low constitutive activity but were not EBNA-2 responsive. The most 5'-deleted -55/+40 LMP1CAT plasmid had moderate constitutive activity and was not EBNA-2 inducible. Either orientation of the -334/+40 LMP1 sequence conferred EBNA-2 responsiveness when positioned upstream of an enhancerless simian virus 40 or herpes simplex virus thymidine kinase (TK) promoter. EBNA-2 and the cis-acting LMP1 DNA were both required to increase TK promoter-initiated mRNA, indicating that the EBNA-2 effect is at the transcriptional level. Further deletion analysis of the EBNA-2-responsive cis-acting element defined a -234/-92 LMP1 DNA fragment which conveyed EBNA-2 responsiveness to the herpes simplex virus TK promoter. The 5' 30 bp between -234 and -205 were essential for EBNA-2 responsiveness. Thus, these experiments define a 142-bp cis-acting element which is sufficient for conveying EBNA-2 responsiveness and an essential 30-bp component of that element. The role of this element in LMP1 and LMP2B expression and its possible role in LMP2A expression are discussed.

Molecular and biological characterization of a herpes simplex virus type 1 (HSV-1) neuroinvasiveness gene.

Pathogenetic studies of herpes simplex virus type 1 (HSV-1) strains ANG and its mouse brain-passaged descendant ANG path revealed no difference in neurovirulence but a significant difference in neuroinvasiveness. Thus, both viruses induced a fatal encephalitis in mice after direct injection into the brain, but only ANG path induced lethal neurologic disease after inoculation on rear footpads. The difference in neuroinvasiveness is not related to the capacity to replicate in mouse neural tissues or mouse cells in general, but is specifically related to virus entry into the peripheral nervous system in the footpad. Marker rescue experiments in which ANG path genes were used to confer neuroinvasiveness on ANG indicated that the gene that codes for glycoprotein D (gD) is responsible for the phenotypic difference. Analyses of the gD genes by dideoxy-sequencing techniques identified a base difference in the coding sequences and predicted that the ANG gD gene codes for alanine (GCC codon) at amino acid position 84 in the open reading frame and the ANG path gD gene codes for glycine (GGC codon) at this site. Using these data, an oligonucleotide probe predicted to be specific for the ANG path gD gene was prepared, and in Southern blot analyses, this probe revealed that neuroinvasiveness-rescued agents had incorporated the base change seen in the ANG path gD gene. We conclude that HSV-1 glycoprotein D functions to effect neuroinvasiveness and we discuss potential mechanisms that may be involved.

Herpes simplex virus type 1 latency-associated transcription plays no role in establishment or maintenance of a latent infection in murine sensory neurons.

Using herpes simplex viruses deleted and restored for the latency-associated transcripts (LATs), we have quantitatively assessed the role of the transcripts in establishment and maintenance of latent infection. Determination of the number of neurons latently infected and the copy number of viral genomes per latently infected ganglion indicated that there is no difference between viruses expressing and not expressing the transcripts. In addition, the amount of viral DNA present in ganglia latently infected with the LAT-negative virus KOS 8117 did not differ from the value for LAT+ counterparts over an 11-month period of analysis. From these results we conclude that LATs play no role in establishment or maintenance of a latent infection with herpes simplex virus type 1. If these transcripts play a role in latency, they must function during the reactivation step.