Overexpression of the Ubiquitin-Specific Protease 7 Resulting from Transfection or Mutations in the ICP0 Binding Site Accelerates Rather than Depresses Herpes Simplex Virus 1 Gene Expression

Earlier studies reported that ICP0, a key regulatory protein encoded by herpes simplex virus 1 (HSV-1), binds ubiquitin-specific protease 7 (USP7). The fundamental conclusion of these studies is that depletion of USP7 destabilized ICP0, that ICP0 mediated the degradation of USP7, and that amino acid substitutions in ICP0 that abolished binding to USP7 significantly impaired the ability of HSV-1 to replicate. We show here that, indeed, depletion of USP7 leads to reduction of ICP0 and that USP7 is degraded in an ICP0-dependent manner. However, overexpression of USP7 or substitution in ICP0 of a single amino acid to abolish binding to USP7 accelerated the accumulation of viral mRNAs and proteins at early times after infection and had no deleterious effect on virus yields. A clue as to why USP7 is degraded emerged from the observation that, notwithstanding the accelerated expression of viral genes, the plaques formed by the mutant virus were very small, implying a defect in virus transmission from cell to cell.     

Immunohistochemical evidence for reorganization of tau in the plaques and tangles in Alzheimer''s dissease

Summary Cytochemical and biochemical techniques have been used to assess the relationship of epitopes on the microtubuleassociated protein, tau, to the cytoskeletal pathology of Alzheimer's disease. The main probes were Tau-1 and Alz-50, two monoclonal antibodies which recognize tau and a potentially related 68kDa protein. Sequential treatment of tissue slices with combinations of the antibodies showed that each blocked the binding of the other to neurofibrillary tangles and neuritic plaques but not to normal axons. Western blot analysis of tau proteins isolated from Alzheimer's disease brains did not reveal such blocking patterns. The issue of steric hindrance affecting antibody binding in tissue sections was addressed by using Alz-50 in combination with Tau-2, another monoclonal antibody recognizing tau on blots and in Alzheimer's disease pathology. Neither antibody blocked the binding of the other to neurofibrillary tangles and neuritic plaques. These data suggest that the Alz-50 and Tau-1 epitopes are selectively organized in the tangles and plaques to be in close proximity which supports the hypothesis that in Alzheimer's disease pathology, tau is modified.     

Tyrosine phosphorylation of the herpes simplex virus type 1 regulatory protein ICP22 and a cellular protein which shares antigenic determinants with ICP22.

At least eight herpes simplex virus type 1 (HSV-1) and five HSV-2 proteins were tyrosine phosphorylated in infected cells. The first viral tyrosine phosphoprotein identified was the HSV-1 regulatory protein ICP22. Also, two novel phosphotyrosine proteins were bound by anti-ICP22 antibodies. H(R22) is a cellular protein, while the F(R10) protein is observed only in HSV-1-infected cells.     

Blocking immune evasion as a novel approach for prevention and treatment of herpes simplex virus infection

Many microorganisms encode immune evasion molecules to escape host defenses. Herpes simplex virus type 1 glycoprotein gC is an immunoevasin that inhibits complement activation by binding complement C3b. gC is expressed on the virus envelope and infected cell surface, which makes gC potentially accessible to blocking antibodies. Mice passively immunized with gC monoclonal antibodies prior to infection were protected against herpes simplex virus challenge only if the gC antibodies blocked C3b binding. Mice treated 1 or 2 days postinfection with gC monoclonal antibodies that block C3b binding had less severe disease than control mice treated with nonimmune immunoglobulin G (IgG). Mice immunized with gC protein produced antibodies that blocked C3b binding to gC. Immunized mice were significantly protected against challenge by wild-type virus, but not against a gC mutant virus lacking the C3b binding domain, suggesting that protection was mediated by antibodies that target the gC immune evasion domain. IgG and complement from subjects immunized with an experimental herpes simplex virus glycoprotein gD vaccine neutralized far more mutant virus defective in immune evasion than wild-type virus, supporting the importance of immune evasion molecules in reducing vaccine potency. These results suggest that it is possible to block immune evasion domains on herpes simplex virus and that this approach has therapeutic potential and may enhance vaccine efficacy.     

The pathology of the neuronal cytoskeleton in Alzheimer's disease.

The two characteristic neuropathological lesions of Alzheimer's disease are the neurofibrillary tangles and the senile plaques. Neurofibrillary tangles are made of abnormal filaments (PHF) accumulating in neurons and mainly composed of a modified form of the microtubule-associated protein tau (PHF-tau). Senile plaques are composed of a cluster of dystrophic neurites surrounding an extracellular deposit of amyloid fibers made of a 42 amino-acid peptide (beta-amyloid peptide). The abnormal filaments contain the complete sequences of the different tau isoforms. The PHF-tau proteins can be distinguished from the normal tau proteins by the presence of several phosphorylated sites. One of these sites is phosphorylated by a calcium-calmodulin-dependent kinase. The relationship between PHF-tau and the cytoskeletal pathology in Alzheimer's disease is further discussed.     

Stimulation of enveloped virus infection by beta-amyloid fibrils

Alzheimer's disease is characterized by deposition of beta-amyloid peptide (Abeta) into plaques in the brain, leading to neuronal toxicity and dementia. Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system can also cause a dementia, and amyloid deposition in the central nervous system is significantly higher in HIV-1-infected individuals compared with uninfected controls. Here we report that Abeta fibrils stimulated, by 5-20-fold, infection of target cells expressing CD4 and an appropriate coreceptor by multiple HIV-1 isolates but did not permit infection of cells lacking these receptors. Abeta enhanced infection at the stage of virus attachment or entry into the cell. Abeta fibrils also stimulated infection by amphotrophic Moloney leukemia virus, herpes simplex virus, and viruses pseudotyped with the envelope glycoprotein of vesicular stomatitis virus. Other synthetic fibril-forming peptides similarly enhanced viral infection and may be useful in gene delivery applications utilizing retroviral vectors. These data suggest that Abeta deposition may increase the vulnerability of the central nervous system to enveloped viral infection and that amyloidogenic peptides could be useful in enhancing gene transfer by enveloped viral vectors.     

Varicella-zoster virus (VZV) open reading frame 10 protein, the homolog of the essential herpes simplex virus protein VP16, is dispensable for VZV replication in vitro.

Varicella-zoster virus (VZV) open reading frame 10 (ORF10) protein in the homolog of the herpes simplex virus type 1 (HSV-1) protein VP16. VZV ORF10 transactivates the VZV IE62 gene and is a tegument protein present in the virion. HSV-1 VP16, a potent transactivator of HSV-1 immediate-early genes and tegument protein, is essential for HSV-1 replication in vitro. To determine whether VZV ORF10 is required for viral replication in vitro, we constructed two VZV mutants which were unable to express ORF10. One mutant had a stop codon after the 61st codon of the ORF10 gene, and the other mutant was deleted for all but the last five codons of the gene. Both VZV mutants grew in cell culture to titers similar to that of the parental virus. To determine whether HSV-1 VP16 alters the growth of VZV, we constructed a VZV mutant in which VP16 was inserted in place of ORF10. Using immune electron microscopy, we found that HSV-1 VP16 was present in the tegument of the recombinant VZV virions. The VZV VP16 substitution mutant produced smaller plaques and grew to a lower titer than parental virus. Thus, VZV ORF10 is not required for growth of the virus in vitro, and substitution of HSV-1 VP16 for VZV ORF10 impairs the growth of VZV.     

Virucidal effect of peppermint oil on the enveloped viruses herpes simplex virus type 1 and type 2 in vitro

The virucidal effect of peppermint oil, the essential oil of Mentha piperita, against herpes simplex virus was examined. The inhibitory activity against herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells using a plaque reduction assay. The 50% inhibitory concentration (IC50) of peppermint oil for herpes simplex virus plaque formation was determined at 0.002% and 0.0008% for HSV-1 and HSV-2, respectively. Peppermint oil exhibited high levels of virucidal activity against HSV-1 and HSV-2 in viral suspension tests. At noncytotoxic concentrations of the oil, plaque formation was significantly reduced by 82% and 92% for HSV-1 and HSV-2, respectively. Higher concentrations of peppermint oil reduced viral titers of both herpesviruses by more than 90%. A clearly time-dependent activity could be demonstrated, after 3 h of incubation of herpes simplex virus with peppermint oil an antiviral activity of about 99% could be demonstrated. In order to determine the mode of antiviral action of the essential oil, peppermint oil was added at different times to the cells or viruses during infection. Both herpesviruses were significantly inhibited when herpes simplex virus was pretreated with the essential oil prior to adsorption. These results indicate that peppermint oil affected the virus before adsorption, but not after penetration into the host cell. Thus this essential oil is capable to exert a direct virucidal effect on HSV. Peppermint oil is also active against an acyclovir resistant strain of HSV-1 (HSV-1-ACV(res)), plaque formation was significantly reduced by 99%. Considering the lipophilic nature of the oil which enables it to penetrate the skin, peppermint oil might be suitable for topical therapeutic use as virucidal agent in recurrent herpes infection.     

Molecular genetics of herpes simplex virus. V. Characterization of a mutant defective in ability to form plaques at low temperatures and in a viral fraction which prevents accumulation of coreless capsids at nuclear pores late in infection.

In herpes simplex virus-infected cells, coreless capsids accumulate at the nuclear pores soon after infection, but subsequently disappear, suggesting that, as in adenovirus-infected cells (S. Dales and Y. Chardonnet, Virology 56:465-483, 1973), the release of viral DNA from nucleocapsids takes place at the nuclear pores. A nonlethal mutant, HSV-1(50B), produced by mutagenesis of HSV DNA fragments and selected for delayed production of plaques at 31 degrees C, accumulated coreless capsids at the nuclear pores late in infection in contrast to wild-type viruses. Recombinants selected for ability to produce plaques at 31 degrees C by marker rescue with digests of herpes simplex virus 2 DNA and selected clone fragments of HSV-1 DNA no longer accumulated empty capsids at nuclear pores late in infection. These results suggest that herpes simplex viruses encode a function which prevents accumulation of coreless capsids at nuclear pores, presumably by preventing uptake, unenvelopment, and DNA release from progeny virus, and indicate that the cold sensitivity of plaque formation and accumulation of coreless capsids might be related or comap in the S component of the genome.     

A cellular function is required for pseudorabies virus envelope glycoprotein processing and virus egress.

The mouse L-cell mutant gro29 is defective for egress of herpes simplex virus type 1 (HSV-1) virions and is significantly reduced in HSV-1 glycoprotein export (B. W. Banfield and F. Tufaro, J. Virol. 64:5716-5729, 1990). In this report, we demonstrate that pseudorabies virus (PRV), a distantly related alphaherpesvirus, shows a distinctive set of defects after infection of gro29 cells. Specifically, we identify defects in the rate and extent of viral glycoprotein export, infectious particle formation, plaque formation, and virus egress. The initial rate of viral glycoprotein synthesis was unaffected in gro29 cells, but the extent of export from the endoplasmic reticulum to the Golgi apparatus was impaired and export through the Golgi apparatus became essentially blocked late in infection. Moreover, by using a secreted variant of a viral membrane protein, we found that export from the Golgi apparatus out of the cell was also defective in gro29 cells. PRV does not form plaques on gro29 monolayers. A low level of infectious virus is formed and released early after infection, but further virus egress is blocked. Taken together, these observations suggest that the gro29 phenotype involves either multiple proteins or a single protein used at multiple steps in viral glycoprotein export and virus egress from cells. Moreover, this host cell protein is required by both HSV and PRV for efficient propagation in infected cells.     

Characterization of Varicella-Zoster virus glycoprotein K (open reading frame 5) and its role in virus growth

Varicella-zoster virus (VZV) is an alphaherpesvirus that is the causative agent of chickenpox and herpes zoster. VZV open reading frame 5 (ORF5) encodes glycoprotein K (gK), which is conserved among alphaherpesviruses. While VZV gK has not been characterized, and its role in viral replication is unknown, homologs of VZV gK in herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) have been well studied. To identify the VZV ORF5 gene product, we raised a polyclonal antibody against a fusion protein of ORF5 codons 25 to 122 with glutathione S-transferase and used it to study the protein in infected cells. A 40,000-molecular-weight protein was detected in cell-free virus by Western blotting. In immunogold electron microscopic studies, VZV gK was in enveloped virions and was evenly distributed in the cytoplasm in infected cells. To determine the function of VZV gK in virus growth, a series of gK deletion mutants were constructed with VZV cosmid DNA derived from the Oka strain. Full and partial deletions in gK prevented viral replication when the gK mutant cosmids were transfected into melanoma cells. Insertion of the HSV-1 (KOS) gK gene into the endogenous VZV gK site did not compensate for the deletion of VZV gK. The replacement of VZV gK at a nonnative AvrII site in the VZV genome restored the phenotypic characteristics of intact recombinant Oka (rOka) virus. Moreover, gK complementing cells transfected with a full gK deletion mutant exhibited viral plaques indistinguishable from those of rOka. Our results are consistent with the studies of gK proteins of HSV-1 and PRV showing that gK is indispensable for viral replication.     

Herpes simplex virus type 1 evades the effects of antibody and complement in vivo

Herpes simplex virus type 1 (HSV-1) encodes a complement-interacting glycoprotein, gC, and an immunoglobulin G (IgG) Fc binding glycoprotein, gE, that mediate immune evasion by affecting multiple aspects of innate and acquired immunity, including interfering with complement components C1q, C3, C5, and properdin and blocking antibody-dependent cellular cytotoxicity. Previous studies evaluated the individual contributions of gC and gE to immune evasion. Experiments in a murine model that examines the combined effects of gC and gE immune evasion on pathogenesis are now reported. Virulence of wild-type HSV-1 is compared with mutant viruses defective in gC-mediated C3 binding, gE-mediated IgG Fc binding, or both immune evasion activities. Eliminating both activities greatly increased susceptibility of HSV-1 to antibody and complement neutralization in vitro and markedly reduced virulence in vivo as measured by disease scores, virus titers, and mortality. Studies with C3 knockout mice indicated that other activities attributed to these glycoproteins, such as gC-mediated virus attachment to heparan sulfate or gE-mediated cell-to-cell spread, do not account for the reduced virulence of mutant viruses. The results support the importance of gC and gE immune evasion in vivo and suggest potential new targets for prevention and treatment of HSV disease.     

The C terminus of the large tegument protein pUL36 contains multiple capsid binding sites that function differently during assembly and cell entry of herpes simplex virus

The largest tegument protein of herpes simplex virus type 1 (HSV1), pUL36, is a multivalent cross-linker between the viral capsids and the tegument and associated membrane proteins during assembly that upon subsequent cell entry releases the incoming capsids from the outer tegument and viral envelope. Here we show that pUL36 was recruited to cytosolic progeny capsids that later colocalized with membrane proteins of herpes simplex virus type 1 (HSV1) and the trans-Golgi network. During cell entry, pUL36 dissociated from viral membrane proteins but remained associated with cytosolic capsids until arrival at the nucleus. HSV1 UL36 mutants lacking C-terminal portions of increasing size expressed truncated pUL36 but could not form plaques. Cytosolic capsids of mutants lacking the C-terminal 735 of the 3,164 amino acid residues accumulated in the cytosol but did not recruit pUL36 or associate with membranes. In contrast, pUL36 lacking only the 167 C-terminal residues bound to cytosolic capsids and subsequently colocalized with viral and host membrane proteins. Progeny virions fused with neighboring cells, but incoming capsids did not retain pUL36, nor could they target the nucleus or initiate HSV1 gene expression. Our data suggest that residues 2430 to 2893 of HSV1 pUL36, containing one binding site for the capsid protein pUL25, are sufficient to recruit pUL36 onto cytosolic capsids during assembly for secondary envelopment, whereas the 167 residues of the very C terminus with the second pUL25 binding site are crucial to maintain pUL36 on incoming capsids during cell entry. Capsids lacking pUL36 are targeted neither to membranes for virus assembly nor to nuclear pores for genome uncoating.     

Herpes simplex virus type 1 and 2 glycoprotein C prevents complement-mediated neutralization induced by natural immunoglobulin M antibody

Glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) binds complement component C3b and protects virus from complement-mediated neutralization. Differences in complement interacting domains exist between gC of HSV-1 (gC1) and HSV-2 (gC2), since the amino terminus of gC1 blocks complement C5 from binding to C3b, while gC2 fails to interfere with this activity. We previously reported that neutralization of HSV-1 gC-null virus by HSV antibody-negative human serum requires activation of C5 but not of downstream components of the classical complement pathway. In this report, we evaluated whether activation of C5 is sufficient to neutralize HSV-2 gC-null virus, or whether formation of the membrane attack complex by C6 to C9 is required for neutralization. We found that activation of the classical complement pathway up to C5 was sufficient to neutralize HSV-2 gC-null virus by HSV antibody-negative human serum. We evaluated the mechanisms by which complement activation occurred in seronegative human serum. Interestingly, natural immunoglobulin M antibodies bound to virus, which triggered activation of C1q and the classical complement pathway. HSV antibody-negative sera obtained from four individuals differed over an approximately 10-fold range in their potency for complement-mediated virus neutralization. These findings indicate that humans differ in the ability of their innate immune systems to neutralize HSV-1 or HSV-2 gC-null virus and that a critical function of gC1 and gC2 is to prevent C5 activation.