A mutation in the DNA polymerase accessory factor of herpes simplex virus 1 restores viral DNA replication in the presence of raltegravir.

UNLABELLED: Previous reports showed that raltegravir, a recently approved antiviral compound that targets HIV integrase, can inhibit the nuclease function of human cytomegalovirus (HCMV terminase) in vitro. In this study, subtoxic levels of raltegravir were shown to inhibit the replication of four different herpesviruses, herpes simplex virus 1 (HSV-1), HSV-2, HCMV, and mouse cytomegalovirus, by 30- to 700-fold, depending on the dose and the virus tested. Southern blotting and quantitative PCR revealed that raltegravir inhibits DNA replication of HSV-1 rather than cleavage of viral DNA. A raltegravir-resistant HSV-1 mutant was generated by repeated passage in the presence of 200 µM raltegravir. The genomic sequence of the resistant virus, designated clone 7, contained mutations in 16 open reading frames. Of these, the mutations F198S in unique long region 15 (UL15; encoding the large terminase subunit), A374V in UL32 (required for DNA cleavage and packaging), V296I in UL42 (encoding the DNA polymerase accessory factor), and A224S in UL54 (encoding ICP27, an important transcriptional regulator) were introduced independently into the wild-type HSV-1(F) genome, and the recombinant viruses were tested for raltegravir resistance. Viruses bearing both the UL15 and UL32 mutations inserted within the genome of the UL42 mutant were also tested. While the UL15, UL32, and UL54 mutant viruses were fully susceptible to raltegravir, any virus bearing the UL42 mutation was as resistant to raltegravir as clone 7. Overall, these results suggest that raltegravir may be a valuable therapeutic agent against herpesviruses and the antiviral activity targets the DNA polymerase accessory factor rather than the nuclease activity of the terminase. IMPORTANCE: This paper shows that raltegravir, the antiretrovirus drug targeting integrase, is effective against various herpesviruses. Drug resistance mapped to the herpesvirus DNA polymerase accessory factor, which was an unexpected finding.

Pharmacological inhibition of VIP signaling enhances antiviral immunity and improves survival in murine cytomegalovirus-infected allogeneic bone marrow transplant recipients.

Cytomegalovirus (CMV) infection following allogeneic bone marrow transplant (allo-BMT) is controlled by donor-derived cellular immunity. Vasoactive intestinal peptide (VIP) suppresses Th1 immunity. We hypothesized that blocking VIP-signaling would enhance anti-CMV immunity in murine recipients of allo-BMT. Recipients were transplanted with bone marrow (BM) and T-cells from major histocompatibility complex (MHC)-mismatched VIP-knockout (KO) or wild-type donors, and treated with 7 daily subcutaneous injections of VIPhyb (peptidic VIP-antagonist) or phosphate-buffered saline (PBS). Genetic and pharmacological blockade of VIP-signaling protected allo-BMT recipients from lethal murine CMV (mCMV) infection, improving survival without increasing graft-versus-host disease. Mice treated with VIPhyb or transplanted with VIP-KO allografts had significantly lower viral loads, increased numbers of mCMV-M45-peptide-MHC-tetramer(+) CD8(+) T-cells, with lower PD-1 expression, and enhanced primary and secondary cellular immune responses after mCMV infection than did PBS-treated mice. These results demonstrate that administration of a VIP antagonist after allo-BMT is a promising safely therapeutic approach to enhance antiviral cellular immunity.

Riboflavin and ultraviolet light for pathogen reduction of murine cytomegalovirus in blood products.

BACKGROUND: Two studies were performed to test the effectiveness of riboflavin and ultraviolet (UV) light treatment (Mirasol PRT, Terumo BCT) against murine cytomegalovirus (MCMV). The first study utilized immune-compromised mice to measure the reduction of cell-free MCMV. A second study used a murine model to evaluate the ability of Mirasol PRT to prevent transfusion-transmitted (TT)-MCMV infection. STUDY DESIGN AND METHODS: Human plasma was inoculated with MCMV and then treated with Mirasol PRT. The viral titer was measured using an infectious dose 50% assay in nude mice. Mice were euthanized on Day 10 posttransfusion, and their spleens were tested for the presence of MCMV DNA using polymerase chain reaction (PCR). Mirasol PRT was also evaluated to determine its effectiveness in preventing TT-MCMV in platelets (PLTs) stored in PLT additive solution. PLTs were inoculated with either cell-associated MCMV or cell-free MCMV and then treated with Mirasol PRT. Mice were transfused with treated or untreated product and were euthanized 14 days posttransfusion. Blood and spleens were assayed for MCMV DNA by real-time-PCR. RESULTS: Using nude mice to titer MCMV, a modest 2.1-log reduction was observed in plasma products after Mirasol PRT treatment. TT-MCMV was not observed in the mouse transfusion model when either cell-free or cell-associated MCMV was treated with Mirasol PRT; MCMV transmission was uniformly observed in mice transfused with untreated PLTs. CONCLUSIONS: These results suggest that using riboflavin and UV light treatment may be able to reduce the occurrence of transmission of human CMV from infectious PLTs and plasma units.

Interferon γ-dependent migration of microglial cells in the retina after systemic cytomegalovirus infection.

Microglial cells are the resident macrophages of the central nervous system and participate in both innate and adaptive immune responses but can also lead to exacerbation of neurodegenerative pathologies after viral infections. Microglia in the outer layers of the retina and the subretinal space are thought to be involved in retinal diseases where low-grade chronic inflammation and oxidative stress play a role. This study investigated the effect of systemic infection with murine cytomegalovirus on the distribution and dynamics of retinal microglia cells. Systemic infection with murine cytomegalovirus elicited a significant increase in the number of microglia in the subretinal space and an accumulation of iris macrophages, along with morphological signs of activation. Interferon γ (IFN-γ)-deficient mice failed to induce changes in microglia distribution. Bone marrow chimera experiments confirmed that microglial cells in the subretinal space were not recruited from the circulating monocyte pool, but rather represented an accumulation of resident microglial cells from within the retina. Our results demonstrate that a systemic viral infection can lead to IFN-γ-mediated accumulation of microglia into the outer retinal layers and offer proof of concept that systemic viral infections alter the ocular microenvironment and therefore, may influence the course of diseases such as macular degeneration, diabetic retinopathy, or autoimmune uveitis, where low-grade inflammation is implicated.

Squalamine as a broad-spectrum systemic antiviral agent with therapeutic potential.

Antiviral compounds that increase the resistance of host tissues represent an attractive class of therapeutic. Here, we show that squalamine, a compound previously isolated from the tissues of the dogfish shark (Squalus acanthias) and the sea lamprey (Petromyzon marinus), exhibits broad-spectrum antiviral activity against human pathogens, which were studied in vitro as well as in vivo. Both RNA- and DNA-enveloped viruses are shown to be susceptible. The proposed mechanism involves the capacity of squalamine, a cationic amphipathic sterol, to neutralize the negative electrostatic surface charge of intracellular membranes in a way that renders the cell less effective in supporting viral replication. Because squalamine can be readily synthesized and has a known safety profile in man, we believe its potential as a broad-spectrum human antiviral agent should be explored.

Blockade of pan-viral propagation by inhibition of host cell PNPT1.

For successful viral propagation within infected cells, the virus needs to overcome the cellular integrated stress response (ISR), triggered during viral infection, which, in turn, inhibits general protein translation. This paper reports a tactic employed by viruses to suppress the ISR by upregulating host cell polyribonucleotide nucleotidyltransferase 1 (PNPT1). The propagation of adenovirus, murine cytomegalovirus and hepatovirus within their respective host cells induces PNPT1 expression. Notably, when PNPT1 is knocked down, the propagation of all three viruses is prevented. Mechanistically, the inhibition of PNPT1 facilitates the relocation of mitochondrial double-stranded RNAs (mt-dsRNAs) to the cytoplasm, where they activate RNA-activated protein kinase (PKR). This activation leads to eukaryotic initiation factor 2α (eIF2α) phosphorylation, resulting in the suppression of translation. Furthermore, by scrutinizing the PNPT1 recognition element and screening 17,728 drugs and bioactive compounds approved by the US Food and Drug Administration, lanatoside C was identified as a potent PNPT1 inhibitor. This compound impedes the propagation of adenovirus, murine cytomegalovirus and hepatovirus, and suppresses production of the severe acute respiratory syndrome coronavirus-2 spike protein. These discoveries shed light on a novel strategy to impede pan-viral propagation by activating the host cell mt-dsRNA-PKR-eIF2α signalling axis.

An Elastin-like Polypeptide-fusion peptide targeting capsid-tegument interface as an antiviral against cytomegalovirus infection.

The tegument protein pp150 of Human Cytomegalovirus (HCMV) is known to be essential for the final stages of virus maturation and mediates its functions by interacting with capsid proteins. Our laboratory has previously identified the critical regions in pp150 important for pp150-capsid interactions and designed peptides similar in sequence to these regions, with a goal to competitively inhibit capsid maturation. Treatment with a specific peptide (PepCR2 or P10) targeted to pp150 conserved region 2 led to a significant reduction in murine CMV (MCMV) growth in cell culture, paving the way for in vivo testing in a mouse model of CMV infection. However, the general pharmacokinetic parameters of peptides, including rapid degradation and limited tissue and cell membrane permeability, pose a challenge to their successful use in vivo. Therefore, we designed a biopolymer-stabilized elastin-like polypeptide (ELP) fusion construct (ELP-P10) to enhance the bioavailability of P10. Antiviral efficacy and cytotoxic effects of ELP-P10 were studied in cell culture, and pharmacokinetics, biodistribution, and antiviral efficacy were studied in a mouse model of CMV infection. ELP-P10 maintained significant antiviral activity in cell culture, and this conjugation significantly enhanced P10 bioavailability in mouse tissues. The fluorescently labeled ELP-P10 accumulated to higher levels in mouse liver and kidneys as compared to the unconjugated P10. Moreover, viral titers from vital organs of MCMV-infected mice indicated a significant reduction of virus load upon ELP-P10 treatment. Therefore, ELP-P10 has the potential to be developed into an effective antiviral against CMV infection.

Cytomegalovirus-induced salivary gland pathology: AREG, FGF8, TNF-α, and IL-6 signal dysregulation and neoplasia.

Mucoepidermoid carcinoma (MEC) is the most common malignant tumor originating in major and minor salivary glands (SGs). Although the precise multifactorial etiology of human SG-MEC is largely unknown, we have recently shown that cytomegalovirus (CMV) is an important component of MEC tumorigenesis. Despite the well-documented overexpression of the EGFR → ERK signaling pathway in SG-MEC, there has been limited to no clinical success with inhibition of this pathway. Using our previously characterized mouse model of CMV-induced SG dysplasia/neoplasia, we report that inhibitors of the EGFR → ERK pathway do not ameliorate or rescue well-established pathology, either singly or in combination, but they do inhibit the evolution of progressive pathogenesis ("disease tolerance") in the face of mounting CMV burden. Failure to rescue SG pathology, suggested a possible increase in the ligand levels of alternative pathways that share cell proliferation and survival effectors (e.g. ERK and PI3K). Here we present evidence of a highly significant upregulation of ligands for the EGFR, FGFR, IL-6R, and TNFR signaling pathways, all of which converge upon the Raf/MEK/ERK amplifier module. This explains our finding that even in the presence of the highest nontoxic dose of an ERK phosphorylation inhibitor, pERK is undiminished. Given the considerable pathway crosstalk, a deep understanding of subversion and dysregulation of the SG interactome by CMV is a priori quite daunting. Circumventing this dilemma, we present evidence that concurrent inhibition of ERK phosphorylation (U0126) and CMV replication (acyclovir) obviates progressive pathogenesis and results in complete SG rescue (tumor regression). These findings provide a mechanistic foundation for potential clinical trials that utilize similar concurrent treatment with extant FDA-approved drugs.

The Synthesis and Anti-Cytomegalovirus Activity of Piperidine-4-Carboxamides.

Treatment options for human cytomegalovirus (CMV) remain limited and are associated with significant adverse effects and the selection of resistant CMV strains in transplant recipients and congenitally infected infants. Although most approved drugs target and inhibit the CMV DNA polymerase, additional agents with distinct mechanisms of action are needed for the treatment and prevention of CMV. In a large high throughput screen using our CMV-luciferase reporter Towne, we identified several unique inhibitors of CMV replication. Here, we synthesize and test in vitro 13 analogs of the original NCGC2955 hit (1). Analogs with no activity against the CMV-luciferase at 10 µM and 30 µM (2-6, 10-14) were removed from further analysis. Three analogs (7-9) inhibited CMV replication in infected human foreskin fibroblasts. The EC50 of (1) was 1.7 ± 0.6 µM and 1.99 ± 0.15 µM, based on luciferase and plaque assay, respectively. Compounds 7, 8, and 9 showed similar activities: the EC50 values of 7 were 0.21 ± 0.06 µM (luciferase) and 0.55 ± 0.06 (plaque), of 8: 0.28 ± 0.06 µM and 0.42 ± 0.07, and of 9: 0.30 ± 0.05 µM (luciferase) and 0.35 ± 0.07 (plaque). The CC50 for 7, 8, and 9 in non-infected human foreskin fibroblasts was > 500µM, yielding a selectivity index of >1500. Compounds 1, 7, and 8 were also tested in CMV-infected primary human hepatocytes and showed a dose-response against CMV by luciferase activity and viral protein expression. None of the active compounds inhibited herpes simplex virus 1 or 2. Compounds 7 and 8 inhibited mouse CMV replication in vitro. Both inhibited CMV at late stages of replication; 7 reduced virus yield at all late time points, although not to the same degree as letermovir. Finally, the activity of analog 8 was additive with newly identified CMV inhibitors (MLS8969, NFU1827, MSL8554, and MSL8091) and with ganciclovir. Further structural activity development should provide promising anti-CMV agents for use in clinical studies.

Effects of allitridin on acute and chronic mouse cytomegalovirus infection.

This study investigated the effects of allitridin on acute and chronic mouse cytomegalovirus (MCMV) infections in vivo. The results demonstrated that allitridin reduced the titers of MCMV in salivary glands, and reductions in viral loads were confirmed by determining viral DNA and RNA levels in susceptible organs during the acute infection phase. Although allitridin did not eliminate MCMV, treatment reduced viral levels and facilitated healing of pathologic lesions in organs, particularly during the chronic infection phase. The results presented in this report suggest that allitridin could act as an effective agent against MCMV infections in vivo.

[Influence of allitridin on transcription, expression and function of IL-12 genes in mice infected by murine cytomegalovirus].

OBJECTIVE: To investigate whether allitridin could interfere with the effects of murine cytomegalovirus (MCMV) infection on the transcription, expression and function of IL-12 genes in order to further explore the mechanism of allitridin against MCMV. METHOD: Sixty mice were randomly divided into allitridin treated group, placebo and blank controls. Allitridin was intra-peritoneal injected to mice in treated group once a day with general dosage (25 mg x kg(-1)) at 24 hours after MCMV infection, and the same dosage of physiological saline were given to placebo and blank groups. Four experimental mice were sacrificed at 3, 5, 7, 10, 14 days after treatment (n = 4 per time point), respectively. The expression of IL-12 p70 and IFN-gamma in supernatant of spleen cell cultures were measured by double-antibody sandwich ELISA, and IL-12 p35 and p40 mRNAs in spleen cells were analyzed by RT-PCR. RESULT: In systemic infection mice, the expression of both IL-12 p70 protein and p35 mRNA significantly increased on day 3 post-infection (pi); then rapidly and markedly decreased on day 5 pi and later. The level of IFN-gamma reached the peak on day 3 pi, then gradually dropped and returned to normal levels during the period of day 10 to 14 pi, and IL-12 p40 mRNA level was persistently and significantly higher after infection. In allitridin treated mice, the levels of IL-12 p70 protein, IL-12 p35 and p40 mRNAs reached the peak on day 3 after treatment (P < 0.05), and then rapidly dropped to the normal levels during the period of 5-14 days. Level of IFN-gamma was also reached the peak on day 3 after treatment; however, it dropped a little on day 5 and then gradually increased and was much higher than those of both placebo and bland controls during the period of day 7 to 14 after treatment (P < 0.01). CONCLUSION: Allitridin could completely correct the disturbance of expression of IL-12 gene caused by MCMV and persistently promote IFN-gamma expression, which was useful for enhancing the specific cellular immune reactions against CMV and clearance of CMV viruses from host. The result suggests another mechanism of allitridin against CMV.

Nedd8-Activating Enzyme Is a Druggable Host Dependency Factor of Human and Mouse Cytomegalovirus.

Human cytomegalovirus causes diseases in individuals with insufficient immunity. Cytomegaloviruses exploit the ubiquitin proteasome pathway to manipulate the proteome of infected cells. The proteasome degrades ubiquitinated proteins. The family of cullin RING ubiquitin ligases (CRL) regulates the stability of numerous important proteins. If the cullin within the CRL is modified with Nedd8 ("neddylated"), the CRL is enzymatically active, while CRLs lacking Nedd8 modifications are inactive. The Nedd8-activating enzyme (NAE) is indispensable for neddylation. By binding to NAE and inhibiting neddylation, the drug MLN4924 (pevonedistat) causes CRL inactivation and stabilization of CRL target proteins. We showed that MLN4924 elicits potent antiviral activity against cytomegaloviruses, suggesting that NAE might be a druggable host dependency factor (HDF). However, MLN4924 is a nucleoside analog related to AMP, and the antiviral activity of MLN4924 may have been influenced by off-target effects in addition to NAE inhibition. To test if NAE is indeed an HDF, we assessed the novel NAE inhibitor TAS4464 and observed potent antiviral activity against mouse and human cytomegalovirus. Additionally, we raised an MLN4924-resistant cell clone and showed that MLN4924 as well as TAS4464 lose their antiviral activity in these cells. Our results indicate that NAE, the neddylation process, and CRLs are druggable HDFs of cytomegaloviruses.

Requirement for natural killer cell-produced interferon gamma in defense against murine cytomegalovirus infection and enhancement of this defense pathway by interleukin 12 administration.

The presence of natural killer (NK) cells contributes to early defense against murine cytomegalovirus (MCMV) infection. Although NK cells can mediate in vivo protection against MCMV, the mechanism by which they do so has not been defined. The studies presented here evaluate cytokine production by NK cells activated during MCMV infection and the role of NK cell-produced cytokines in early in vivo antiviral defenses. Experiments with normal C57BL/6, T cell-deficient C57BL/6 nude, and severe combined immunodeficient mice lacking T and B cells demonstrated that both interferon gamma (IFN-gamma) and tumor necrosis factor (TNF) production were induced at early times after infection with MCMV. Conditioned media samples prepared with cells from these mice, on day 2 after infection, produced 11-43 pg/million cells of IFN-gamma and 12-19 pg/million cells of TNF as evaluated by specific protein enzyme-linked immunosorbent assays. Studies in the NK- and T cell-deficient mouse line, E26, in mice that had been depleted in vivo of NK cells by treatment with antibodies eliminating NK cells, anti-asialo ganglio-N-tetraosylceramide or anti-NK1.1, and with populations of cells that had been depleted of NK cells by complement treatment with the anti-NK cell antibody, SW3A4, demonstrated that NK cells were solely responsible for the IFN-gamma but were not required for TNF production. The in vivo absence of NK cells was accompanied by increased viral hepatitis and viral replication in both immunocompetent and immunodeficient mice, as well as decreased survival time of immunodeficient mice. In vivo treatments with antibodies neutralizing IFN-gamma demonstrated that this factor contributed to the NK cell-mediated antiviral defense and reduced the measured parameters of viral defense to levels indistinguishable from those observed in NK cell-deficient mice. These effects appeared to be independent of cytolytic activity, as NK cells isolated from anti-IFN-gamma-treated mice mediated killing at levels comparable to those observed in control-treated mice. The consequences of interleukin 12 (IL-12) administration, a known potent inducer of IFN-gamma production by NK cells, were evaluated in MCMV-infected mice. Low IL-12 doses, i.e., 1 ng/d, increased NK cell cytotoxicity and IFN-gamma production up to twofold and resulted in improved antiviral status; virus-induced hepatitis was decreased as much as fivefold, and viral burdens were decreased to levels below detection.(ABSTRACT TRUNCATED AT 400 WORDS)

Polyinosinic: Polycytidylic Acid and Murine Cytomegalovirus Modulate Expression of Murine IL-10 and IL-21 in White Adipose Tissue.

White adipose tissue (WAT) produces interleukin-10 and other immune suppressors in response to pathogen-associated molecular patterns (PAMPs). It also homes a subset of B-cells specialized in the production of IL-10, referred to as regulatory B-cells. We investigated whether viral stimuli, polyinosinic: polycytidylic acid (poly(I:C)) or whole replicative murine cytomegalovirus (MCMV), could stimulate the expression of IL-10 in murine WAT using in vivo and ex vivo approaches. Our results showed that in vivo responses to systemic administration of poly(I:C) resulted in high levels of endogenously-produced IL-10 and IL-21 in WAT. In ex vivo WAT explants, a subset of B-cells increased their endogenous IL-10 expression in response to poly(I:C). Finally, MCMV replication in WAT explants resulted in decreased IL-10 levels, opposite to the effect seen with poly(I:C). Moreover, downregulation of IL-10 correlated with relatively lower number of Bregs. To our knowledge, this is the first report of IL-10 expression by WAT and WAT-associated B-cells in response to viral stimuli.

Amphipathic DNA polymers exhibit antiviral activity against systemic murine Cytomegalovirus infection.

BACKGROUND: Phosphorothioated oligonucleotides (PS-ONs) have a sequence-independent, broad spectrum antiviral activity as amphipathic polymers (APs) and exhibit potent in vitro antiviral activity against a broad spectrum of herpesviruses: HSV-1, HSV-2, HCMV, VZV, EBV, and HHV-6A/B, and in vivo activity in a murine microbiocide model of genital HSV-2 infection. The activity of these agents against animal cytomegalovirus (CMV) infections in vitro and in vivo was therefore investigated. RESULTS: In vitro, a 40 mer degenerate AP (REP 9) inhibited both murine CMV (MCMV) and guinea pig CMV (GPCMV) with an IC50 of 0.045 microM and 0.16 microM, respectively, and a 40 mer poly C AP (REP 9C) inhibited MCMV with an IC50 of 0.05 microM. Addition of REP 9 to plaque assays during the first two hours of infection inhibited 78% of plaque formation whereas addition of REP 9 after 10 hours of infection did not significantly reduce the number of plaques, indicating that REP 9 antiviral activity against MCMV occurs at early times after infection. In a murine model of CMV infection, systemic treatment for 5 days significantly reduced virus replication in the spleens and livers of infected mice compared to saline-treated control mice. REP 9 and REP 9C were administered intraperitoneally for 5 consecutive days at 10 mg/kg, starting 2 days prior to MCMV infection. Splenomegaly was observed in infected mice treated with REP 9 but not in control mice or in REP 9 treated, uninfected mice, consistent with mild CpG-like activity. When REP 9C (which lacks CpG motifs) was compared to REP 9, it exhibited comparable antiviral activity as REP 9 but was not associated with splenomegaly. This suggests that the direct antiviral activity of APs is the predominant therapeutic mechanism in vivo. Moreover, REP 9C, which is acid stable, was effective when administered orally in combination with known permeation enhancers. CONCLUSION: These studies indicate that APs exhibit potent, well tolerated antiviral activity against CMV infection in vivo and represent a new class of broad spectrum anti-herpetic agents.

In vivo proof-of-concept for two experimental antiviral drugs, both directed to cellular targets, using a murine cytomegalovirus model.

Infections with the human cytomegalovirus (HCMV) cause serious medical problems including organ rejection and congenital infection. Treatment of HCMV infections with currently available medication targeting viral enzymes is often accompanied with severe side effects and the occurrence of drug-resistant viruses. This demands novel therapeutical approaches like targeting genetically stable host cell proteins that are crucial for virus replication. Although numerous experimental drugs with promising in vitro efficacy have been identified, the lack of available data in animal models limits their potential for further clinical development. Recently, we described the very strong in vitro antiherpesviral activity of the NF-κB inhibitor TF27 and the CDK7 inhibitor LDC4297 at low nanomolar concentrations. In the present study, we present first data for the in vivo efficacy of both experimental drugs using an established cytomegalovirus animal model (murine CMV replication in immunodefective Rag -/- mice). The main findings of this study are (i) a strong inhibitory potency against beta- and gamma-herpesviruses of both compounds in vitro, (ii) even more important, a pronounced anticytomegaloviral activity also exerted in vivo, that resulted from (iii) a restriction of viral replication to the site of infection, thus preventing organ dissemination, (iv) in the absence of major compound-associated adverse events. Thus, we provide evidence for a strong antiviral potency in vivo and proof-of-concept for both drugs, which may encourage their further drug development, possibly including pharmacologically optimized derivatives, for a potential use in future antiherpesviral treatment.

A viral kinase counteracts in vivo restriction of murine cytomegalovirus by SAMHD1.

The deoxynucleotide triphosphate (dNTP) hydrolase SAMHD1 inhibits retroviruses in non-dividing myeloid cells. Although antiviral activity towards DNA viruses has also been demonstrated, the role of SAMHD1 during cytomegalovirus (CMV) infection remains unclear. To determine the impact of SAMHD1 on the replication of CMV, we used murine CMV (MCMV) to infect a previously established SAMHD1 knockout mouse model and found that SAMHD1 inhibits the replication of MCMV in vivo. By comparing the replication of MCMV in vitro in myeloid cells and fibroblasts from SAMHD1-knockout and control mice, we found that the viral kinase M97 counteracts SAMHD1 after infection by phosphorylating the regulatory residue threonine 603. The phosphorylation of SAMHD1 in infected cells correlated with a reduced level of dNTP hydrolase activity and the loss of viral restriction. Together, we demonstrate that SAMHD1 acts as a restriction factor in vivo and we identify the M97-mediated phosphorylation of SAMHD1 as a previously undescribed viral countermeasure.

Anticytomegalovirus Peptides Point to New Insights for CMV Entry Mechanisms and the Limitations of In Vitro Screenings.

Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that can cause severe disease following in utero exposure, during primary infection, or latent virus reactivation in immunocompromised populations. These complications lead to a 1- to 2-billion-dollar economic burden, making vaccine development and/or alternative treatments a high priority. Current treatments for HCMV include nucleoside analogues such as ganciclovir (GCV), foscarnet, and cidofovir. Recently, letermovir, a terminase complex inhibitor, was approved for prophylaxis after stem cell transplantation. These treatments have unwanted side effects, and HCMV is becoming resistant to them. Therefore, we sought to develop an alternative treatment that targets a different stage in viral infection. Currently, small antiviral peptides are being investigated as anti-influenza and anti-HIV treatments. We have developed heparan sulfate-binding peptides as tools for preventing CMV infections. These peptides are highly effective at stopping infection of fibroblasts with in vitro-derived HCMV and murine cytomegalovirus (MCMV). However, they do not prevent MCMV infection in vivo Interestingly, these peptides inhibit infectivity of in vivo-derived CMVs, albeit not as well as tissue culture-grown CMVs. We further demonstrate that this class of heparan sulfate-binding peptides is incapable of inhibiting MCMV cell-to-cell spread, which is independent of heparan sulfate usage. These data indicate that inhibition of CMV infection can be achieved using synthetic polybasic peptides, but cell-to-cell spread and in vivo-grown CMVs require further investigation to design appropriate anti-CMV peptides.IMPORTANCE In the absence of an effective vaccine to prevent HCMV infections, alternative interventions must be developed. Prevention of viral entry into susceptible cells is an attractive alternative strategy. Here we report that heparan sulfate-binding peptides effectively inhibit entry into fibroblasts of in vitro-derived CMVs and partially inhibit in vivo-derived CMVs. This includes the inhibition of urine-derived HCMV (uCMV), which is highly resistant to antibody neutralization. While these antiviral peptides are highly effective at inhibiting cell-free virus, they do not inhibit MCMV cell-to-cell spread. This underscores the need to understand the mechanism of cell-to-cell spread and differences between in vivo-derived versus in vitro-derived CMV entry to effectively prevent CMV's spread.

Establishment of a cell-based assay for screening of compounds inhibiting very early events in the cytomegalovirus replication cycle and characterization of a compound identified using the assay.

To simplify the detection of infectious human cytomegalovirus (HCMV), we generated a cell line that produced luciferase in a dose-dependent manner upon HCMV infection. Using this cell line, we identified anti-HCMV compounds from a diverse library of 9,600 compounds. One of them, 1-(3,5-dichloro-4-pyridyl)piperidine-4-carboxamide (DPPC), was effective against HCMV (Towne strain) infection of human lung fibroblast cells at a 50% effective concentration of 2.5 microM. DPPC also inhibited the growth of clinical HCMV isolates and guinea pig and mouse cytomegaloviruses. Experiments using various time frames for treatment of the cells with DPPC demonstrated that DPPC was effective during the first 24 h after HCMV infection. DPPC treatment decreased not only viral DNA replication but also IE1 and IE2 expression at mRNA and protein levels in the HCMV-infected cells. However, DPPC did not inhibit the attachment of HCMV particles to the cell surface. DPPC is a unique compound that targets the very early phase of cytomegalovirus infection, probably by disrupting a pathway that is important after viral entry but before immediate-early gene expression.

Effect of oral treatment with (S)-HPMPA, HDP-(S)-HPMPA or ODE-(S)-HPMPA on replication of murine cytomegalovirus (MCMV) or human cytomegalovirus (HCMV) in animal models.

We utilized BALB/c mice infected with murine CMV (MCMV) or severe combined immunodeficient (SCID) mice implanted with human fetal tissue and infected with HCMV to determine the efficacy of (S)-9-[3-hydroxy-2-(phophonomethoxy)propyl]adenine ((S)-HPMPA), hexadecyloxypropyl-(S)-HPMPA (HDP-(S)-HPMPA) or octadecyloxyethyl-(S)-HPMPA (ODE-(S)-HPMPA). In MCMV-infected BALB/c mice, oral HDP-(S)-HPMPA at 30 mg/kg significantly reduced mortality when started 24-48 h post inoculation. In the experimental HCMV infection, oral administration of vehicle or 10mg/kg of (S)-HPMPA, HDP-(S)-HPMPA or ODE-(S)-HPMPA was initiated 24h after infection and continued for 28 consecutive days. Cidofovir (CDV), at 20mg/kg given i.p., was used as a positive control. HDP-(S)-HPMPA or ODE-(S)-HPMPA significantly reduced viral replication compared to vehicle-treated mice, while oral (S)-HPMPA was ineffective.