Viral Induced Protein Aggregation: A Mechanism of Immune Evasion.

Various intrinsic and extrinsic factors can interfere with the process of protein folding, resulting in protein aggregates. Usually, cells prevent the formation of aggregates or degrade them to prevent the cytotoxic effects they may cause. However, during viral infection, the formation of aggregates may serve as a cellular defense mechanism. On the other hand, some viruses are able to exploit the process of aggregate formation and removal to promote their replication or evade the immune response. This review article summarizes the process of cellular protein aggregation and gives examples of how different viruses exploit it. Particular emphasis is placed on the ribonucleotide reductases of herpesviruses and how their additional non-canonical functions in viral immune evasion are closely linked to protein aggregation.

Therapeutic Mucosal Vaccination of Herpes Simplex Virus 2-Infected Guinea Pigs with Ribonucleotide Reductase 2 (RR2) Protein Boosts Antiviral Neutralizing Antibodies and Local Tissue-Resident CD4+ and CD8+ TRM Cells Associated with Protection against Recurrent Genital Herpes.

Reactivation of herpes simplex virus 2 (HSV-2) from latency causes viral shedding that develops into recurrent genital lesions. The immune mechanisms of protection against recurrent genital herpes remain to be fully elucidated. In this preclinical study, we investigated the protective therapeutic efficacy, in the guinea pig model of recurrent genital herpes, of subunit vaccine candidates that were based on eight recombinantly expressed HSV-2 envelope and tegument proteins. These viral protein antigens (Ags) were rationally selected for their ability to recall strong CD4+ and CD8+ T-cell responses from naturally "protected" asymptomatic individuals, who, despite being infected, never develop any recurrent herpetic disease. Out of the eight HSV-2 proteins, the envelope glycoprotein D (gD), the tegument protein VP22 (encoded by the UL49 gene), and ribonucleotide reductase subunit 2 protein (RR2; encoded by the UL40 gene) produced significant protection against recurrent genital herpes. The RR2 protein, delivered either intramuscularly or intravaginally with CpG and alum adjuvants, (i) boosted the highest neutralizing antibodies, which appear to cross-react with both gB and gD, and (ii) enhanced the numbers of functional gamma interferon (IFN-γ)-producing CRTAM+ CFSE+ CD4+ and CRTAM+ CFSE+ CD8+ TRM cells, which express low levels of PD-1 and TIM-3 exhaustion markers and were localized to healed sites of the vaginal mucocutaneous (VM) tissues. The strong B- and T-cell immunogenicity of the RR2 protein was associated with a significant decrease in virus shedding and a reduction in both the severity and frequency of recurrent genital herpes lesions. In vivo depletion of either CD4+ or CD8+ T cells significantly abrogated the protection. Taken together, these preclinical results provide new insights into the immune mechanisms of protection against recurrent genital herpes and promote the tegument RR2 protein as a viable candidate Ag to be incorporated in future genital herpes therapeutic mucosal vaccines.IMPORTANCE Recurrent genital herpes is one of the most common sexually transmitted diseases, with a global prevalence of HSV-2 infection predicted to be over 536 million worldwide. Despite the availability of many intervention strategies, such as sexual behavior education, barrier methods, and the costly antiviral drug treatments, eliminating or at least reducing recurrent genital herpes remains a challenge. Currently, no FDA-approved therapeutic vaccines are available. In this preclinical study, we investigated the immunogenicity and protective efficacy, in the guinea pig model of recurrent genital herpes, of subunit vaccine candidates that were based on eight recombinantly expressed herpes envelope and tegument proteins. We discovered that similar to the dl5-29 vaccine, based on a replication-defective HSV-2 mutant virus, which has been recently tested in clinical trials, the RR2 protein-based subunit vaccine elicited a significant reduction in virus shedding and a decrease in both the severity and frequency of recurrent genital herpes sores. This protection correlated with an increase in numbers of functional tissue-resident IFN-γ+ CRTAM+ CFSE+ CD4+ and IFN-γ+ CRTAM+ CFSE+ CD8+ TRM cells that infiltrate healed sites of the vaginal tissues. Our study sheds new light on the role of TRM cells in protection against recurrent genital herpes and promotes the RR2-based subunit therapeutic vaccine to be tested in the clinic.

Inhibition of proinflammatory and innate immune signaling pathways by a cytomegalovirus RIP1-interacting protein.

TNFalpha is an important cytokine in antimicrobial immunity and inflammation. The receptor-interacting protein RIP1 is an essential component of the TNF receptor 1 signaling pathway that mediates the activation of NF-kappaB, MAPKs, and programmed cell death. It also transduces signals derived from Toll-like receptors and intracellular sensors of DNA damage and double-stranded RNA. Here, we show that the murine CMV M45 protein binds to RIP1 and inhibits TNFalpha-induced activation of NF-kappaB, p38 MAPK, and caspase-independent cell death. M45 also inhibited NF-kappaB activation upon stimulation of Toll-like receptor 3 and ubiquitination of RIP1, which is required for NF-kappaB activation. Hence, M45 functions as a viral inhibitor of RIP1-mediated signaling. The results presented here reveal a mechanism of viral immune subversion and demonstrate how a viral protein can simultaneously block proinflammatory and innate immune signaling pathways by interacting with a central mediator molecule.

The efficacy of antigen processing is critical for protection against cytomegalovirus disease in the presence of viral immune evasion proteins.

Cytomegaloviruses (CMVs) code for immunoevasins, glycoproteins that are specifically dedicated to interfere with the presentation of antigenic peptides to CD8 T cells. Nonetheless, the biological outcome is not an immune evasion of the virus, since CD8 T cells can control CMV infection even when immunoevasins are expressed. Here, we compare the processing of a protective and a nonprotective epitope derived from the same viral protein, the antiapoptotic protein M45 in the murine model. The data provide evidence to conclude that protection against CMVs critically depends on antigenic peptides generated in an amount sufficient to exhaust the inhibitory capacity of immunoevasins.

Herpesviruses induce aggregation and selective autophagy of host signalling proteins NEMO and RIPK1 as an immune-evasion mechanism.

Viruses manipulate cellular signalling by inducing the degradation of crucial signal transducers, usually via the ubiquitin-proteasome pathway. Here, we show that the murine cytomegalovirus (Murid herpesvirus 1) M45 protein induces the degradation of two cellular signalling proteins, the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) essential modulator (NEMO) and the receptor-interacting protein kinase 1 (RIPK1), via a different mechanism: it induces their sequestration as insoluble protein aggregates and subsequently facilitates their degradation by autophagy. Aggregation of target proteins requires a distinct sequence motif in M45, which we termed 'induced protein aggregation motif'. In a second step, M45 recruits the retromer component vacuolar protein sorting 26B (VPS26B) and the microtubule-associated protein light chain 3 (LC3)-interacting adaptor protein TBC1D5 to facilitate degradation of aggregates by selective autophagy. The induced protein aggregation motif is conserved in M45-homologous proteins of several human herpesviruses, including herpes simplex virus, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, but is only partially conserved in the human cytomegalovirus UL45 protein. We further show that the HSV-1 ICP6 protein induces RIPK1 aggregation and degradation in a similar fashion to M45. These data suggest that induced protein aggregation combined with selective autophagy of aggregates (aggrephagy) represents a conserved viral immune-evasion mechanism.

Quantitation of G0 and G1 phase cells in primary carcinomas. Antibody to M1 subunit of ribonucleotide reductase shows G1 phase restriction point block.

Human cancers have an apparent low growth fraction, the bulk of cells presumed to being out of cycle in a G0 quiescent state due to the inability in the past to distinguish G0 from G1 cells. The allosteric M1 subunit of ribonucleotide reductase (M1-RR) is constitutively expressed by cycling cells (i.e., G1, S, G2-M). It is acquired during transition from G0 to G1, lost during exit to G0 and thus distinguishes G0 from G1 cells. To estimate the proportion of G0 and G1 cells in primary human breast (n = 5) and colorectal (n = 12) adenocarcinomas, we used both analytical DNA flow cytometry (ADFC) and immunoperoxidase staining of sections with the monoclonal antibody to M1-RR (MAb M1-RR). ADFC of fresh tumors revealed a low percentage of cells in the S phase (4.0 +/- 3.4%) but immunoperoxidase staining for M1-RR revealed an unexpectedly high proportion of positive cells (52.4 +/- 12.7%) in the G1, S, G2-M phases indicating a high G1 content of primary human tumors. Thus, human cancers are blocked in transition in G1 and are not predominantly in a G0 or quiescent differentiated state. This block was interpreted to mean that human cancers are responding to putative regulatory events at a restriction point in the G1 phase, such as relative growth factor deficiency, density inhibition, antiproliferative cytokines, or gene products. Using flow cytometry for both DNA and M1-RR content we found that human colon cancer cell lines arrest in the G1 but not G0 phase upon serum deprivation or density inhibition. Similarly, human breast cancer cell lines are arrested in G1 but not G0 phase by medroxyprogesterone acetate (MPA) or tamoxifen exposure. These findings match our in situ observations, and support the concept of a restriction point block in primary human tumors.

Identification of the gene for the yeast ribonucleotide reductase small subunit and its inducibility by methyl methanesulfonate.

We have identified, cloned, and sequenced the gene for the small subunit of ribonucleotide diphosphate reductase of Saccharomyces cerevisiae. The protein and its transcript are induced about 10-fold by the alkylating agent methyl methanesulfonate, a result which suggests that the gene is induced by DNA damage.

Deletion of F4L (ribonucleotide reductase) in vaccinia virus produces a selective oncolytic virus and promotes anti-tumor immunity with superior safety in bladder cancer models.

Bladder cancer has a recurrence rate of up to 80% and many patients require multiple treatments that often fail, eventually leading to disease progression. In particular, standard of care for high-grade disease, Bacillus Calmette-Guérin (BCG), fails in 30% of patients. We have generated a novel oncolytic vaccinia virus (VACV) by mutating the F4L gene that encodes the virus homolog of the cell-cycle-regulated small subunit of ribonucleotide reductase (RRM2). The F4L-deleted VACVs are highly attenuated in normal tissues, and since cancer cells commonly express elevated RRM2 levels, have tumor-selective replication and cell killing. These F4L-deleted VACVs replicated selectively in immune-competent rat AY-27 and xenografted human RT112-luc orthotopic bladder cancer models, causing significant tumor regression or complete ablation with no toxicity. It was also observed that rats cured of AY-27 tumors by VACV treatment developed anti-tumor immunity as evidenced by tumor rejection upon challenge and by ex vivo cytotoxic T-lymphocyte assays. Finally, F4L-deleted VACVs replicated in primary human bladder cancer explants. Our findings demonstrate the enhanced safety and selectivity of F4L-deleted VACVs, with application as a promising therapy for patients with BCG-refractory cancers and immune dysregulation.

Comparative immunogenicity of M. hyopneumoniae NrdF encoded in different expression systems delivered orally via attenuated S. typhimurium aroA in mice.

The Mycoplasma hyopneumoniae ribonucleotide reductase R2 subunit (NrdF) gene fragment was cloned into eukaryotic and prokaryotic expression vectors and its immunogenicity evaluated in mice immunized orally with attenuated Salmonella typhimurium aroA CS332 harboring either of the recombinant expression plasmids. We found that NrdF is highly conserved among M. hyopneumoniae strains. The immunogenicity of NrdF was examined by analyzing antibody responses in sera and lung washes, and the cell-mediated immune (CMI) response was assessed by determining the INF-gamma level produced by splenocytes upon in vitro stimulation with NrdF antigen. S. typhimurium expressing NrdF encoded by the prokaryotic expression plasmid (pTrcNrdF) failed to elicit an NrdF-specific serum or secretory antibody response, and IFN-gamma was not produced. Similarly, S. typhimurium carrying the eukaryotic recombinant plasmid encoding NrdF (pcNrdF) did not induce a serum or secretory antibody response, but did elicit significant NrdF-specific IFN-gamma production, indicating induction of a CMI response. However, analysis of immune responses against the live vector S. typhimurium aroA CS332 showed a serum IgG response but no mucosal IgA response in spite of its efficient invasiveness in vitro. In the present study we show that the DNA vaccine encoding the M. hyopneumoniae antigen delivered orally via a live attenuated S. typhimurium aroA can induce a cell-mediated immune response. We also indicate that different live bacterial vaccine carriers may have an influence on the type of the immune response induced.

Hypothesis: the MHC-restricted T-cell receptor as a structure with two multistate allosteric combining sites.

This paper presents a dual-recognition model of the T-cell receptor that has been constructed to account for the phenomenon of MHC restriction as well as the paradoxical ability of T-cells to be both multispecific and precisely specific at the same time. In our model the combining sites for antigen and MHC are not independent as in classical dual-recognition models, but interact with each other by an allosteric mechanism. We envision a flexible receptor with combining sites for antigen and MHC that are capable of existing in a multitude of distinct complementarity states. MHC and antigen molecules act as allosteric effectors such that one ligand perturbs the conformation and therefore the specificity of the site for the other ligand. An essential feature of the model is that different MHC determinants induce different conformations at the anti-antigen site. In this way the receptor acquires multiple specificities. Within a particular complementarity state, precise recognition results from the requirement that antigen and MHC exhibit positive cooperativity in their binding to the T-cell receptor. Positive cooperativity is also the basis for MHC restriction. Reaction mechanisms are presented which describe the requirement that antigen and MHC both induce conformational changes in order to generate high-affinity binding to either ligand. As a precedent for the multistate allosteric receptor model, we discuss the properties of allosteric enzymes, especially ribonucleotide reductase, whose properties are analogous to those we have postulated for the T-cell receptor. Also discussed is the possibility that molecules such as Ly2, L3T4 and the Mls antigen, which have been found to play a role in antigen recognition, function as affinity-enhancing allosteric effectors that interact with the constant portion of the T-cell receptor.

Immunologic characterization of herpes simplex virus type 2 antigens ICP10 and ICSP11/12.

Infected cell protein 10 (ICP10) or antigen 4 (Ag4) and infected cell-specific protein 11/12 (ICSP11/12) have been suggested as specific antigenic markers for cervical carcinoma. Experiments were designed to determine whether ICP10 and ICSP11/12 are distinct antigens and to determine the cellular localization of ICP10. Results indicate that an apparent 160 kdalton (kDa) protein analyzed by 8.5% polyacrylamide gels (= 144 kDa protein analyzed by 7.0% polyacrylamide gels) was detected in HSV-2-infected but not mock-infected extracts. This protein is an early virus-induced protein appearing 2-4 h after HSV-2 infection, and it was synthesized in the presence of successive blocks with cycloheximide and actinomycin D. These properties are characteristic for ICP10 (Ag4), thus establishing the identity of the 160 kDa/144 kDa protein as ICP10. Furthermore, Western blot analyses indicated that ICP10 and ICSP11/12 are distinct antigens recognized by antibodies in sera from immune rabbit or human cervical carcinoma patients. In addition, monoclonal antibodies to the HSV-2-induced ribonucleotide reductase were reactive with ICP10. Antibodies in sera from rabbits immunized against ICP10 and monoclonal antibodies to the HSV-2-induced ribonucleotide reductase were reactive with antigens on the plasma membrane surface of HSV-2-infected cells. Also, the reactivity of monoclonal antibodies with these antigens was blocked by the rabbit antibodies based on immunofluorescence analyses. These data provide evidence that ICP10 is antigenically distinct from ICSP11/12, and that ICP10 is present on the plasma membrane of HSV-2-infected cells. Also, our data confirm and extend the tentative identification of ICP10 with the HSV-2-induced ribonucleotide reductase recently suggested by Bacchetti et al. (J. Virol. 49, 591-593, 1984).

Differences in the properties of mammalian ribonucleotide reductase toward its substrates.

These studies, using three different reagents, show that the substrate properties of ribonucleotide reductase are specific but can be variable depending upon the nature of the interaction of the reagent with the holoenzyme or the individual subunit. Etheno-CDP, which acts as a competitive inhibitor with respect to CDP, interacts with the active site of the holoenzyme. This interaction was the result of rather tight structural requirements as epsilon-ADP did not result in a similar level of inhibition of either CDP or ADP reductase activities. The YL 1/2 antibody which binds very tightly to the NHI subunit has a much greater effect on CDP reductase activity than ADP reductase activity. The nonapeptide that corresponds to the C-terminus amino acid sequence of the NHI subunit and which binds to the EB subunit and aborts the formation of the NHI-EB active complex has a greater effect on ADP reductase activity than on CDP reductase activity. The use of reagents such as these can be helpful in dissecting the subtle but important differences in the substrate properties of mammalian ribonucleotide reductase.

A monoclonal antibody against HSV type 1 ribonucleotide reductase cross-reacts with the P0 protein of peripheral nerve myelin.

An epitope on peripheral nerve myelin was detected by the use of a mouse monoclonal antibody directed against the 38 kDa subunit of herpes simplex virus (HSV) type 1 ribonucleotide reductase. Immunohistochemistry showed reactivity solely in PNS myelin. In nerve roots there was a sharp border in transitional zones to the negative CNS myelin. The immunoreactivity was found in rat, guinea pig, bovine and human peripheral nerves. Western blot analysis of peripheral nerve myelin as well as purified P0 revealed a distinctly stained band corresponding to a molecular weight of approximately 29 kDa. The present finding of a shared antigenic determinant between HSV ribonucleotide reductase and peripheral nerve P0 may be of pathogenetic relevance in virus induced demyelinating diseases in the peripheral nervous system.

A double-blind study of the efficacy and safety of the ICP10deltaPK vaccine against recurrent genital HSV-2 infections.

A randomized double-blind trial to evaluate the safety of a novel recombinant virus, ICP10deltaPK, for reduction or prevention of recurrent herpes simplex virus type 2 (HSV-2) infection was carried out in public hospitals in Mexico City. Persons having a minimum of 5 documented herpetic recurrences in the previous year were randomized for vaccination. Patients were examined within 72 hours of lesion occurrence. If accepted into the study, the patient was inoculated subcutaneously in the upper deltoid muscle area at days 7, 17, and 28 after initiation of lesion occurrence. Recurrences were recorded by patient diary and physician examination. During the observation period (extending from 10 to 180 days after the last booster dose), recurrences in the vaccine (V) group were prevented completely in 37.5% of the patients, whereas in the placebo (P) group, 100% of the patients had at least one recurrence (P = .068). Vaccinated patients had fewer recurrences (V, 1.58; P, 3.13 [P = .028]). The mean number of illness days was 10 for the vaccine group and 18 for the placebo group (P = .028). Further studies to evaluate this vaccine and its dosimetry for the treatment of genital herpes infections appear warranted.

Protection of shrimp Penaeus monodon from WSSV infection using antisense constructs.

White spot syndrome caused by white spot syndrome virus (WSSV) is one of the most threatening diseases of shrimp culture industry. Previous studies have successfully demonstrated the use of DNA- and RNA-based vaccines to protect WSSV infection in shrimp. In the present study, we have explored the protective efficacy of antisense constructs directed against WSSV proteins, VP24, and VP28, thymidylate synthase (TS), and ribonucleotide reductase-2 (RR2) under the control of endogenous shrimp histone-3 (H3) or penaedin (Pn) promoter. Several antisense constructs were generated by inserting VP24 (pH3-VP24, pPn-VP24), VP28 (pH3-VP28, pPn-VP28), TS (pH3-TS, pPn-TS), and RR2 (pH3-RR2) in antisense orientation. These constructs were tested for their protective potential in WSSV infected cell cultures, and their effect on reduction of the viral load was assessed. A robust reduction in WSSV copy number was observed upon transfection of antisense constructs in hemocyte cultures derived from Penaeus monodon and Scylla serrata. When tested in vivo, antisense constructs offered a strong protection in WSSV challenged P. monodon. Constructs expressing antisense VP24 and VP28 provided the best protection (up to 90 % survivability) with a corresponding decrease in the viral load. Our work demonstrates that shrimp treated with antisense constructs present an efficient control strategy for combating WSSV infection in shrimp aquaculture.

Ribonucleotide reductase small subunit M2B prognoses better survival in colorectal cancer.

Ribonucleotide reductase subunit RRM2B (p53R2) has been reported to suppress invasion and metastasis in colorectal cancer (CRC). Here, we report that high levels of RRM2B expression are correlated with markedly better survival in CRC patients. In a fluorescence-labeled orthotopic mouse xenograft model, we confirmed that overexpression of RRM2B in nonmetastatic CRC cells prevented lung and/or liver metastasis, relative to control cells that did metastasize. Clinical outcome studies were conducted on a training set with 103 CRCs and a validation set with 220 CRCs. All participants underwent surgery with periodic follow-up to determine survivability. A newly developed specific RRM2B antibody was employed to carry out immunohistochemistry for determining RRM2B expression levels on tissue arrays. In the training set, the Kaplan-Meier and multivariate Cox analysis revealed that RRM2B is associated with better survival of CRCs, especially in stage IV patients (HR = 0.40; 95% CI = 0.18-0.86, P = 0.016). In the validation set, RRM2B was negatively related to tumor invasion (OR = 0.45, 95% CI = 0.19-0.99, P = 0.040) and lymph node involvement (OR = 0.48, 95% CI = 0.25-0.92, P = 0.026). Furthermore, elevated expression of RRM2B was associated with better prognosis in this set as determined by multivariate analyses (HR = 0.48, 95% CI = 0.26-0.91, P = 0.030). Further investigations revealed that RRM2B was correlated with better survival of CRCs with advanced stage III and IV tumors rather than earlier stage I and II tumors. Taken together, our findings establish that RRM2B suppresses invasiveness of cancer cells and that its expression is associated with a better survival prognosis for CRC patients.

Production of a monoclonal antibody against the hRRM2 subunit of ribonucleotide reductase and immunohistochemistry study of human cancer tissues.

We report production of a monoclonal antibody against the hRRM2 subunit of ribonucleotide reductase and immunohistochemistry (IHC) staining of human cancer tissues available in paraffin block. BALB/c mice were immunized with purified hRRM2 protein, and splenocytes from these mice were fused with mice myeloma cell lines by using standard hybridoma production techniques. Resulting hybridomas producing anti-hRRM2 antibodies were screened by enzyme-linked immunosorbent assay (ELISA). The specificity was determined by limiting serial dilutions. Clones were chosen for antibody production based on their activities on paraffin-embedded human tissues. They were then isotyped and shown to produce immunoglobulin M (IgM) antibodies against hRRM2. Using these antibodies, we performed Western blot on oropharyngeal KB cancer cell lines and immunohistochemistry staining of available paraffin-embedded cancer tissues. Interestingly, cancer tissues stained positive with the anti-hRRM2 antibody but not normal tissues. Colon, stomach, liver, lung, pancreatic, and breast cancer had the strongest staining. No staining was identified on astrocytoma, mesothelioma, or myeloma. Our findings were validated with data from reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrating overexpression of hRRM2 in breast cancer tissues compared to matched noncancer tissues. We propose that IHC with this monoclonal anti-hRRM2 antibody may be useful for ribonucleotide reductase research and as a biomarker for tumorgenesis.

Neutralization of herpes simplex virus ribonucleotide reductase activity by an oligopeptide-induced antiserum directed against subunit H2.

Herpes simplex virus type 1 ribonucleotide reductase is associated with two polypeptides of apparent molecular weights 136,000 and 38,000. The two polypeptides form a tight complex and, therefore, are often coprecipitated by monoclonal antibodies. We report here that immunoglobulins G purified from polyclonal rabbit antisera (P9) raised against a nonapeptide corresponding to the carboxy terminus of the 38,000-dalton polypeptide specifically neutralize the herpes simplex virus ribonucleotide reductase activity. We suggest that the P9 immunoglobulin G neutralizes the reductase activity by impairing the association of the two subunits (H1 and H2) of the enzyme.

Is ribonucleotide reductase the transforming function of herpes simplex virus 2?

Transformation of cells by herpes simplex virus 2 (HSV-2) can be induced by the BglII C (0.43-0.58 map units) or N (0.58-0.625) fragments of the viral genome. Sequences partially overlapping both fragments (0.566-0.602) encode two 3' coterminal mRNAs; these in turn direct the synthesis of two related polypeptides of molecular weight 140,000 (140K) and 35K (refs 4, 7), which may be involved in transformation. Recently, a temperature-sensitive (ts) mutation affecting HSV-induced ribonucleotide reductase has been mapped within this common region (B.M. Dutia, personal communication). We have partially purified the induced reductase and raised a rabbit antiserum to it which inhibits the enzyme activity and immunoprecipitates from infected cells a 144K polypeptide and minor species including a 38K polypeptide. Here we show that a monoclonal antibody to the putative transforming proteins competes with the rabbit serum for the 144K and 38K antigens and also immunoprecipitates specifically the induced reductase activity. These results suggest a possible role for ribonucleotide reductase in HSV-2-induced transformation.