Herpes Simplex Virus Type 1 Infected Cells Research Articles

Conversion of trichosanthin-induced CD95 (Fas) type I into type II apoptotic signaling during Herpes simplex virus infection

Trichosanthin (TCS) is a type I ribosome-inactivating protein with wide spectrum of pharmacological activities. It inhibits human immunodeficiency virus type 1 (HIV-1) and Herpes simplex virus type 1 (HSV-1) replication but the mechanism is not clear. From a previous study, TCS was found to be more cytotoxic to HIV-1 infected cells than uninfected cells. Similar finding was confirmed with HSV-1 in the present study. TCS induced cell death in HEp-2 cells and the EC50 was 24.64μg/mL. When the same experiment was performed in HSV-1 infected HEp-2 cells, the EC50 decreased to 3.01μg/mL. TCS appeared to cause more death and apoptosis in viral infected cells. This study explored plausible mechanism with respect to the apoptosis signal pathways. In uninfected cells, TCS induced CD95 (Fas)-mediated and caspase-8-dependent type I apoptosis. When cells were infected with HSV-1, apoptosis induced by TCS clearly switched to a more potent type II pathway. This involved mitochondrial depolarization and caspase-9 activation. The major evidences arose from studying the individual signals of the two apoptosis pathways in infected and uninfected cells. In addition, over expression of Bcl-2, which mainly affected the type II pathway reduced TCS induced apoptosis mostly in infected cells. This further demonstrated that the type II pathway was operating in infected cells. The reason for the switching is not entirely clear but it is well known that viral infection affects signal pathways especially those related to apoptosis. In conclusion, TCS selectively induces more apoptosis in HSV-1 infected cells than uninfected cells. The consequence of infection switches the TCS-induced apoptosis pathway from a CD95 (Fas) dependent type I to a more potent type II pathway mediated by mitochondrial depolarization and caspase-9 activation.

Analysis of the cell cycle regulatory protein (E2F1) after infection of cultured cells with bovine herpesvirus 1 (BHV-1) or herpes simplex virus type 1 (HSV-1)

The E2F family of cellular transcription factors controls cell cycle progression and cell death. During cell cycle progression, activated cyclin-dependent kinases phosphorylate the retinoblastoma (Rb) protein, causing the release and activation of E2F family members. Previous studies demonstrated that bovine herpes virus 1 (BHV-1) productive infection increases E2F1 protein levels, the bICP0 early promoter is activated more than 100 fold by E2F1 or E2F2, and silencing E2F1 reduced the efficiency of productive infection. In this study, the effect of herpes simplex virus type 1 (HSV-1) productive infection on E2F protein levels and regulation of E2F dependent transcription was compared to BHV-1 infection in the same permissive cell line, rabbit skin (RS) cells. Silencing E2F1 with a specific siRNA reduced HSV-1 productive infection approximately 10 fold in RS cells, and total E2F1 protein levels increased during productive infection. In contrast to RS cells infected with BHV-1, a fraction of total E2F1 protein was localized to the cytoplasm in HSV-1 infected RS cells. Furthermore, E2F1 did not efficiently trans-activate the HSV-1 ICP0 or ICP4 promoter. When RS cells were transfected with an E2F reporter construct or the cyclin D1 promoter and then infected with BHV-1, promoter activity increased after infection. In contrast, HSV-1 infection of RS cells had little effect on E2F dependent transcription and cyclin D1 promoter activity was reduced. In summary, these studies indicated that silencing E2F1 reduced the efficiency of HSV-1 and BHV-1 productive infection. However, only BHV-1 productive infection induced E2F dependent transcription.

Antiserum to the recombinant truncated VP22 protein of herpes simplex virus type 1 that also recognizes full-length VP22

The herpes simplex virus type 1 (HSV-1) tegument protein VP22 encoded by the UL49 gene is essential for HSV-1 infection. However, its precise functions in the virus life cycle are unknown. A relatively important tool for disclosing these functions is an antiserum specifically detecting VP22 in the infected cell. To this end, a recombinant truncated VP22 protein consisting of C-terminal 45 aa fused to EYFP (enhanced yellow fluorescent protein) and His-tag was expressed in Escherichia coli, purified by the Ni2+-NTA affinity chromatography, and used for the preparation of antiserum in rabbits. Western blot and immunofluorescence assay showed that this antiserum specifically detected purified truncated VP22 as well as full-length VP22 in the HSV-1 infected cells. These results indicate that the prepared antiserum could serve as a valuable tool for further studies of VP22 functions.

Herpes simplex virus type 1 ICP27 protein: Its expression, purification and specific antiserum production

Herpes simplex virus type 1 (HSV-1) is the causative agent of cold sores and other more serious diseases. HSV-1 infected-cell protein 27 (ICP27) is an immediate-early regulatory phosphoprotein homologous to gene products identified in all classes of herpesviruses so far. To raise the antiserum to ICP27 for further characterization of its biological function, the ICP27 gene was cloned into the pET-28a (+) vector, then ICP27 protein was expressed in E. coli and purified by nickel-nitrilotriacetic acid (Ni(2+)-NTA) affinity resin column, finally the purified protein was used to raise antiserum. Western blot analysis demonstrated that the antiserum recognized the recombinant protein, and the antiserum was able to probe the ICP27 in HSV-1 infected cells with high specificity by immunofluorescence assay (IFA). Therefore, the specific antiserum will provide a valuable tool for further studies investigating ICP27's biological function during HSV-1 infection.

Herpes Simplex Virus type 1 infection disrupts the ATR‐mediated damage response

Herpes Simplex Virus type 1 (HSV‐1) is a large DNA virus that replicates in the nucleus of the host cell. HSV‐1 infection results in the activation of the ATM mediated damage response but not ATR. Since activation of ATM in uninfected cells generally results in the activation of ATR, we undertook the current study to determine how HSV‐1 infection disrupts the ATR mediated damage response. Preliminary data using phospho‐specific antibodies indicate that RPA and Chk1, which usually are targets of ATR, are not phosphorylated in infected cells. Using immunofluorescence we have shown that RPA and ATRIP are localized to the sites of viral DNA replication and that ATR and ATRIP do not colocalize in infected cells like they do during a normal damage response. We next wanted to see if we could activate the ATR pathway in infected cells by using hydroxyurea (HU). HU is a potent inhibitor of both cellular and viral DNA replication and will result in stalled replication forks and phosphorylation of the ATR substrate Chk1 in uninfected cells. Consistent with our previous data, Chk1 is not phosphorylated in response to HU in HSV‐1 infected cells. Taken together these results indicate that components of the ATR signaling pathway are present in viral replication compartments but that ATR signaling itself is disabled, perhaps by uncoupling ATR from ATRIP. This work was supported by Public Health Service grants AI21747 and AI69136.

Expression, purification of herpes simplex virus type 1 UL4 protein, and production and characterization of UL4 polyclonal antibody

The herpes simplex virus type 1 (HSV-1) UL4 protein is a late protein encoded by the UL4 gene. To date, the function of this protein is poorly understood. To aid further investigation of the function of this protein, the UL4 gene was cloned into the vector pET28a (+) to express His-tagged UL4 protein in Escherichia coli. The recombinant fusion protein was purified from inclusion body by histidine selected nickel affinity chromatography under denaturing conditions. After refolding, the purified recombinant protein was used to produce anti-UL4 polyclonal antibody. Western blot analysis demonstrated that the polyclonal sera could recognize the purified UL4 protein specifically, and in the immunofluorescence assay, the antibody was able to probe the UL4 protein with a punctate staining in HSV-1 infected cells.

Proteomic analysis of cells in the early stages of herpes simplex virus type‐1 infection reveals widespread changes in the host cell proteome

During infection by herpes simplex virus type-1 (HSV-1) the host cell undergoes widespread changes in gene expression and morphology in response to viral replication and release. However, relatively little is known about the specific proteome changes that occur during the early stages of HSV-1 replication prior to the global damaging effects of virion maturation and egress. To investigate pathways that may be activated or utilised during the early stages of HSV-1 replication, 2-DE and LC-MS/MS were used to identify cellular proteome changes at 6 h post infection. Comparative analysis of multiple gels representing whole cell extracts from mock- and HSV-1-infected HEp-2 cells revealed a total of 103 protein spot changes. Of these, 63 were up-regulated and 40 down-regulated in response to infection. Changes in selected candidate proteins were verified by Western blot analysis and their respective cellular localisations analysed by confocal microscopy. We have identified differential regulation and modification of proteins with key roles in diverse cellular pathways, including DNA replication, chromatin remodelling, mRNA stability and the ER stress response. This work represents the first global comparative analysis of HSV-1 infected cells and provides an important insight into host cell proteome changes during the early stages of HSV-1 infection.

The HSV-1 tegument protein pUL46 associates with cellular membranes and viral capsids

The molecular mechanisms responsible for the addition of tegument proteins into nascent herpesvirus particles are poorly understood. To better understand the tegumentation process of herpes simplex virus type 1 (HSV-1) virions, we initiated studies that showed the tegument protein pUL46 (VP11/12) has a similar cellular localization to the membrane-associated tegument protein VP22. Using membrane flotation analysis we found that pUL46 associates with membranes in both the presence and absence of other HSV-1 proteins. However, when purified virions were stripped of their envelope, the majority of pUL46 was found to associate with the capsid fraction. This strong affinity of pUL46 for capsids was confirmed by an in vitro capsid pull-down assay in which purified pUL46-GST was able to interact specifically with capsids purified from the nuclear fraction of HSV-1 infected cells. These results suggest that pUL46 displays a dynamic interaction between cellular membranes and capsids.

Identification of a protein encoded in the herpes simplex virus type 1 latency associated transcript promoter region

Herpes simplex virus type 1 (HSV-1) establishes a lifelong latency in the neurons of its host. Sporadically, the latent virus reactivates and spreads back to the original site of infection and causes recrudescent diseases. The only gene actively transcribed during neuronal latency is the latency associated transcript ( LAT) gene. Several transcripts have been detected in the important LAT promoter region. However, no polypeptides coded by these transcripts are known. In this communication, we reported the cloning, sequencing, and characterization of a transcript immediately upstream of LAT. We designated this gene UOL (Upstream of LAT). The UOL RNA is polyadenylated, expressed as a late gene in infected cells, transcribed in the same direction as LAT, and contains an open reading frame (ORF) capable of encoding a protein of 96 amino acids with a predicted molecular mass of 11 kDa. The UOL transcript contains 466 nucleotides in length. The 5′ end of the UOL transcript starts at nucleotide 118,266 and the 3′ end of the UOL transcript ends at nucleotide 118,731 based on the published 17syn+ genomic sequence. The UOL protein was detected in infected cell lysates by immunoprecipitation using an antibody raised against UOL ORF synthetic peptide. More importantly, sera from mice infected with wild-type HSV-1 but not sera from mice infected with a mutant with the UOL region deleted recognized the UOL ORF, expressed in Escherichia coli, on Western blots. These results suggest that a UOL protein is in HSV-1 infected tissue culture cells and in mice infected with HSV-1.

Effect of HSV-2 infection on the expression of HPV 16 genes in CaSki cells.

Human papillomaviruses (HPVs) have been proposed to be the most important etiological factors for cervical cancer although different agents may act in conjunction. Herpes simplex virus type 2 (HSV-2) infection is considered as a possible cofactor to malignant transformation. To examine the influence of HSV-2 infection on the HPV genes expression, CaSki cells bearing 60 to 600 copies of HPV-16 DNA per cell were used as a model system. Twenty hours post HSV-2 infection the mRNA transcripts for HPV-16 early (E1, E2 and E6) and late (L1) genes were analysed by RT-PCR assay. Results indicated that the level of transcription of E1, E2 and E6 genes was up to 3-fold enhanced in HSV-2 infected CaSki cells suggesting that HSV-2 infection could increase the risk of cervical cancer by overexpression of both HPV regulatory and oncogenic genes.

A novel apoptotic interaction between HSV-1 and human corneal epithelial cells

Purpose. Herpes simplex virus type 1 (HSV-1) infects the cornea possibly causing blindness. The specific mechanisms of herpetic keratitis are unclear. We aimed to investigate whether HSV-1 would up- or down-regulate the apoptotic pathway of human corneal epithelial (HCE) cells. Methods. HSV-1 infection of HCE and Vero cells was demonstrated (immunofluorescence) and apoptotic gene expression was quantified (ribonuclease protection assay). Caspase 8 protein activity (colorimetric assay) was quantified and compared to caspase 8 mRNA amounts from RPA experiments. The apoptotic index of HSV-1 infected HCE and Vero cells (apoptotic index = % of apoptotic cells in infected samples/mock treated samples) was obtained and compared to gene expression. Results. A down-regulation in apoptotic gene expression was observed in HSV-1 infected HCE cells in contrast to Vero cells (infected and mock treated). Caspase 8 protein levels mirrored caspase 8 mRNA levels in HSV-1 infected HCE cells. The apoptotic index also supports this down-regulation. HSV-1 infected human corneal epithelial cells and Vero cells at similar rates. Conclusions. HSV-1 down-regulates the apoptotic pathway of human corneal epithelial cells. This down-regulation of apoptotic gene expression seems to be cell specific. Also infectivity is excluded in playing a role in regulation of the apoptotic pathway because HSV-1 replicated at similar rates in HCE and Vero cells.

Temporal Morphogenesis of Herpes Simplex Virus Type 1-Infected and Brefeldin A-Treated Human Fibroblasts

Insights in the herpesvirus-cell interactions are of general cell biology interest, especially to studies of intracellular transport, and of considerable significance in the efforts to generate drugs, vaccines, and gene therapy. However, the pathway of virus particle egress and maturation is a contentious issue. The intracellular transport was inhibited in cultured herpes simplex virus type 1 (HSV-1) infected human fibroblasts by brefeldin A (BFA). The virus-cell interactions including the viral envelopment, transport of HSV-1 virions, and transport of viral glycoprotein D (gD-1) and glycoprotein C (gC-1) were studied by titration assay, immunoblot, immunofluorescence light microscopy, and immunogold electron microscopy of cryosections. gD-1 and gC-1 were synthesized and normally transported to the plasma membranes of untreated HSV-1 infected host cells. BFA (1 microg/ml medium) effectively blocked the transport of the glycoproteins to the plasma membranes and affected the tubulin and vimentin of the cytoskeleton. Viral particles and glycoproteins accumulated in the perinuclear space and the endoplasmic reticulum of BFA treated cells. Withdrawal of BFA influence up to 9 hr resulted in restored tubulin and vimentin, transport of glycoproteins to the plasma membranes, and steady release of infectious viral particles to the extracellular space superior to the cellular assembly of new virions. The ultrastructural data presented support that the primary envelopment of viral particles occur at the nuclear membranes containing immature glycoproteins followed by multiple de-envelopments and re-envelopments of the virions during the transport and maturation in the endoplasmic reticulum and the Golgi complex. BFA-induced changes include the cytoskeleton with significant effect on HSV-1 maturation and egress. The data support a multiple-step envelopment of HSV-1 in a common pathway of glycoprotein synthesis and virion egress.

The effects of cell passages on the cell morphology and the outcome of herpes simplex virus type 1 infection

Because cell cultures are essential in biological research which involves the analysis of virus morphogenesis, this study focused on examining the significance of cell passages. Human embryonic lung fibroblasts (MRC-5) at passage (P) 27 were seeded twice a week to P 32, P 40, and P 48, when just at confluence and then infected with herpes simplex virus type 1 (HSV-1). The structure of the non-virus-infected (MOCK) and HSV-1 infected cells, the amount of cellular infectious virus particles and the capability to express HSV-1 glycoproteins C (gC-1) and D (gD-1) were investigated by phase-contrast and immunofluorescence light microscopy, immunogold cryosection EM, plaque assays, immunoblots, and total protein assays. Modified cell structure including fragmentation of tubulin fibers were visible in MOCK from P 38 onwards. The quantity of vimentin remained unchanged while actin accumulated and β-tubulin decreased in HSV-1 infected late P cells compared to early P cultures. Cells of high P counts contained significantly fewer infectious virus particles, very likely of lower virulence, and their expression of gC-1 and gD-1 were concordantly reduced. These observations indicate that the number of cell P must be considered in order to reproduce results of cell biology and viral morphogenesis. The MRC-5 cells ought not to be passaged more than ten times beyond P 27 in the laboratory.

Natural killer (NK) cells prevent virus production in cell culture.

Natural killer (NK) cells (CD3-/CD16+/CD56+ lymphocytes) play an important role in early immune defense against viral infection, a fact which is of prime significance for heavily immunosuppressed patients after bone marrow transplantation. In this study we demonstrate that NK cells preferentially lyse human colon adenocarcinoma (Colo-205) tumor cells infected with herpes simplex virus type 1 (HSV-1) and vaccinia virus (VV) and autologous T cells infected with VV. This phenomenon was assessed by the viral infectious center (IC) method and compared with the results obtained by means of the standard 51Cr-release assay. Using the IC assay, we found that NK cells lyse virus infected cells at an early stage of infection, thereby preventing viral dissemination to neighboring cells. 51Cr-release assay verified by propidium iodide (PI) penetration showed that the early effects of NK mediated anti-viral activity are not the result of membrane damage. The effect of NK cells on HSV-1 infected Colo-205 cells appears to be independent of the level of expression of major histocompatibility complex (MHC) class I molecules while the killing of autologous VV-infected T cells correlates with a reduction in MHC class I expression. Our results suggest that additional factors besides MHC play a role in the regulation of NK cell-mediated lysis of virus infected cells. This may be of clinical importance in patients who are heavily immunosuppressed after bone marrow transplantation.

Evaluation of a fully automated glycoprotein G-2 based assay for the detection of HSV-2 specific IgG antibodies in serum and plasma.

Ranking after infections with Chlamydia trachomatis and human papillomavirus, genital herpesvirus is the third most common sexually transmitted disease. The majority of recurrent genital herpes infections are caused by herpes simplex virus type-2 (HSV-2). Seroepidemiological studies on the prevalence of HSV-2 specific IgG antibodies are especially important to study the impact of this infection among risk groups. To evaluate the sensitivity and specificity of the Cobas Core HSV-2 IgG specific assay (available for research use only), that can be run on the Cobas Core fully automated immuno-analyzer. The Cobas Core HSV-2 specific IgG EIA is based on macro-beads coated with affinity purified glycoprotein G-2 antigen from HSV-2 infected cells. The Cobas Core HSV-2 IgG specific assay was compared with the Chiron rapid immunoblot assay (RIBA), the Gull enzyme-linked immunosorbent assay (EIA) and the Centocor EIA. The respective assays were tested, using 1219 serum samples, from 612 females and 607 males attending the outpatient clinic for sexually transmitted diseases of the Erasmus Medical Center Rotterdam (EMCR). The consensus value, obtained by a concordant result with three out of four assays, demonstrated 350 positive samples (28.7%), 851 negative samples (69.8%) and 18 (1.5%) serum samples with a discordant result. The overall agreement of the Cobas Core HSV-2 EIA against the consensus value was 95.8% and the sensitivity and specificity proved to be 100 and 97.1% respectively. The results obtained with the Cobas Core HSV-2 EIA indicate that this is a useful, specific and sensitive assay for the detection of HSV-2 specific IgG antibodies in serum. The advantage of the Cobas Core HVS-2 EIA compared to the other assays, is that this assay can be performed in a fully automated process.

Physical and functional interactions between herpes simplex virus immediate-early proteins ICP4 and ICP27.

The ordered expression of herpes simplex virus type 1 (HSV-1) genes, during the course of a productive infection, requires the action of the virus immediate-early regulatory proteins. Using a protein interaction assay, we demonstrate specific in vitro protein-protein interactions between ICP4 and ICP27, two immediate-early proteins of HSV-1 that are essential for virus replication. We map multiple points of contact between these proteins. Furthermore, by coimmunoprecipitation experiments, we demonstrate the following. (i) ICP4-ICP27 complexes are present in extracts from HSV-1 infected cells. (ii) ICP27 binds preferentially to less modified forms of ICP4, a protein that is extensively modified posttranslationally. We also demonstrate, by performing electrophoretic mobility shift assays and supershifts with monoclonal antibodies to ICP4 or ICP27, that both proteins are present in a DNA-protein complex with a noncanonical ICP4 binding site present in the HSV thymidine kinase (TK) gene. ICP4, in extracts from cells infected with ICP27-deficient viruses, is impaired in its ability to form complexes with the TK site but not with the canonical site from the alpha4 gene. However, ICP4 is able to form complexes with the TK probe, in the absence of ICP27, when overproduced in mammalian cells or expressed in bacteria. These data suggest that the inability of ICP4 from infected cell extracts to bind the TK probe in the absence of ICP27 does not reflect a requirement for the physical presence of ICP27 in the complex. Rather, they imply that ICP27 is likely to modulate the DNA binding activity of ICP4 by affecting its posttranslational modification status. Therefore, we propose that ICP27, in addition to its established role as a posttranscriptional regulator of virus gene expression, may also modulate transcription either through direct or indirect interactions with HSV regulatory regions, or through its ability to modulate the DNA binding activity of ICP4.

抗ＨＳＶ活性を有するアンチセンス・オリゴＤＮＡの安定性およびウイルス感染細胞における動態の検討

We have synthesized antisense phosphorothioate oligodeoxynucleotide (S-ODN) tageted 3' splice junction of immediate early (IE) pre-mRNA 4/5 in herpes simplex virus type 1 (HSV-1) and evaluated their antiviral activities as a novel anti-herpetic agent. The antisense S-ODN completely protected the permissive Vero cells from a cytopathic effect of HSV-1 at a lower concentration range of 0.78 to 1.56 microM in a serum-free assay system, whereas its sequence including mismatced bases of S-ODN and the sense sequence of S-ODN exhibited the protective effect at higher concentrations (=> 50 microM). The results indicated that the antisense S-ODNs targeted 3' splice junction inhibit viral growth in a sequence-specific manner. To get more informations for the usage as a therapeutic agent, the biological stability and the intracellular distribution of the antisense S-ODN were examined. In the nuclease degradation test, the antisense S-ODN had a half life of 5 to 7 hrs in culture medium containing 10% fetal calf serum at 37°C. The similar antisense phosphodiester (D) ODN was completely degraded within 60 mins. The S-ODN was shown to be considerablely more stable to nuclease degradation than D-ODN. In the same condition, the antiviral activity of the antisense S-ODN was 8 times less than that in the serum-free culture system. No antivial effect of the antisense D-ODN was detected at any concentration even in the serum-free culture system. In HSV-1 infected cells, a uniform distribution of S-ODN was observed both in nuclei and in cyto plasm at 12-hr incubation. On the other hand, in non-infected cells, S-ODN was localized into lysosome-like vesicles and such localization of S-ODN continued on and after 12-hr incubation. This indicates that a critical disadvantage of intracellular delivery to target site might be solved by viral infection. These findings suggest that the antisense S-ODN may be sufficiently worth developing as an antiherpetic therapeutic agent, and that some more devices for the improvement of nuclease-resistance and intracellular permeability could make the antisense S-ODN more effective against HSV-1 infection.

Characterization of the US8.5 protein of herpes simplex virus.

In a previous study a novel gene designated as US8.5 was identified in the unique short component of the herpes simplex virus type 1 (HSV1) genome. Epitope tagging experiments suggested the existence of a protein encoded by this gene in HSV1 infected cells. To further analyze the US8.5 gene product and function, a rabbit polyclonal antiserum was raised against a recombinant beta-galactosidase-US8.5 fusion protein expressed in E. coli. This antiserum reacted specifically with a 19 kDa protein in HSV1(F) infected cells as shown by immunoblotting and immunoprecipitation experiments. In addition, using the same antiserum a 21 kDa protein was detected in lysates from cells infected with HSV2(G), which was most likely the HSV2 homolog of US8.5. Kinetic studies indicated that US8.5 is expressed as gamma 1 gene. In addition, the US8.5 protein was immunoprecipitated with the anti-US8.5 serum from 32P-labeled lysates of Vero cells infected with HSV1, demonstrating that the protein is phosphorylated. Immunofluorescence studies localized the US8.5 protein to the nucleoli of HSV1 infected cells.

The cell-coded polypeptide U90 increased by herpes simplex virus type 2 infection induces Fos and DNA synthesis.

Lytic infection with herpes simplex virus type 2 (HSV-2) induces synthesis of a cell-coded protein of molecular mass 90 kDa, termed U90. U90, whose expression is specific in tumour cells is, in addition, excreted from he cell. To determine if the function of U90 could be advantageous for the virus the protein was purified from the medium of HSV-2 infected cells, shown to be similar to the internalized form and its effect on cell growth studied. Addition of U90 to quiescent fibroblast cells stimulated teh expression of Fos, the product of the cellular transcription factor c-fos and increased the incorporation of [3H]thymidine into DNA, factors which may facilitate HSV-2 replication. However, U90 might also induce abnormal cells such as those in precancerous lesions to proliferate.

Inhibition of herpes simplex virus replication by antisense oligo-2'-O-methylribonucleoside methylphosphonates.

Antisense oligonucleoside methylphosphonates complementary to the 12 nucleotides found at the intron/exon junction of the splice acceptor site of herpes simplex virus type 1 (HSV-1) immediate early mRNAs 4 and 5 were synthesized. The methylphosphonate oligomers contained either 2'-deoxyribose nucleosides, d-OMPs, or 2'O-methylribose nucleosides, mr-OMPs. At 37 degrees C, the affinity of the mr-OMP for a complementary 12-mer RNA target was approximately four times higher than that of the corresponding d-OMP as measured by a constant activity gel electrophoresis mobility shift assay. An mr-OMP whose sequence contained two mismatched bases did not bind to the RNA target under these conditions. The mr-OMP also showed improved ability to inhibit HSV-1 replication in HSV-1 infected Vero cells in culture. Thus the IC50 of the mr-OMP was five times less than that of the d-OMP. No inhibition was observed by the mismatched mr-OMP, and no inhibition of herpes simplex virus type 2 (HSV-2) replication was observed with any of the oligomers. These results demonstrate a direct correlation between oligomer binding affinity and antisense activity in cell culture and suggest that oligo-2'-O-methylribonucleoside methylphosphonates are promising candidates for development of effective antisense reagents.