Phosphorothioate-Modified Oligodeoxynucleotides Inhibit Human Cytomegalovirus Replication by Blocking Virus Entry

Abstract

Studies in animal models have provided evidence that Toll-like receptor 9 (TLR9) agonists, such as synthetic oligodeoxynucleotides (ODNs) that contain immunostimulatory deoxycytidyl-deoxyguanosine (CpG) motifs (CpG ODNs), protect against a wide range of viral pathogens. This antiviral activity has been suggested to be indirect and secondary to CpG-induced cytokines and inflammatory responses triggered through TLR9 activation. However, few studies have addressed the potential of CpG ODNs as direct antiviral agents. Here, we report on the ability of some CpG ODNs to directly suppress, almost completely, human cytomegalovirus (HCMV) replication in both primary fibroblasts and endothelial cells. Murine CMV replication was inhibited as well, whereas no inhibition was observed for herpes simplex virus type 1, adenovirus, or vesicular stomatitis virus. The antiviral activity of these ODNs was significantly reduced when they were added after virus adsorption, indicating that their action may be primarily targeted to the very early phases of the HCMV cycle. In fact, the B-class prototype CpG ODN 2006 effectively prevented the nuclear localization of pp65 and input viral DNA, which suggests that it inhibits HCMV entry. Moreover, a CpG 2006 control, ODN 2137 without CpG motifs, also showed a potent inhibitory activity on the HCMV entry phase, indicating that the anticytomegaloviral activity is independent of the CpG motif. In contrast, a phosphodiester version of CpG 2006 showed reduced antiviral activity, indicating that the inhibitory activity is dependent on the phosphorothioate backbone of the ODN. These results suggest that this yet-unrecognized activity of CpG ODNs may be of interest in the development of novel anticytomegaloviral molecules.

Phosphorothioate-Modified TLR9 Ligands Protect Cancer Cells against TRAIL-Induced Apoptosis

“…binding inhibition. This phenomenon could be extended to the inhibition of virus entry, as it has been recently shown that human cytomegalovirus, hepatitis C virus, and HIV entry are blocked by phosphorothioate-modified ODNs, regardless of CpG motifs (33)(34)(35)(36). One could therefore take advantage of this to design antiviral vaccines that target virus entry.…”

Peptide-derivatized dendrimers inhibit human cytomegalovirus infection by blocking virus binding to cell surface heparan sulfate

“…The cells and supernatants from the antiviral assay were then harvested and disrupted by sonication. The extent of virus replication was subsequently assessed by titrating the infectivity of the supernatants of cell suspensions on either HELFs (for HCMV, adenovirus and VSV) or NIH 3T3 cells (for MCMV) as previously described (Luganini et al, 2008a). Plaques were microscopically counted and the mean plaque counts for each drug concentration were expressed as a percentage of the mean plaque count of the control virus.…”

“…Real-time quantitative reverse transcription-PCR (RT-PCR) was performed on an Mx 3000 P apparatus (Stratagene) using SYBR Green as a non-specific PCR product fluorescence label as previously described (Caposio et al, 2007b;Luganini et al, 2008a). After HCMV infection and cell treatment, total cellular RNA was isolated using the Eurozol reagent (Euroclone Ltd., United Kingdom) and RNA samples (1 g) were then retrotranscribed at 42°C for 60 min in PCR buffer (1.5 mM MgCl2) containing 5 M random primers, 0.5 mM deoxynucleoside triphosphates, and 100 U Moloney murine leukemia virus reverse transcriptase (Ambion) in a final volume of 20 l. Reverse-transcribed cDNAs (2 l), or water as the control, were then amplified in duplicate using the Brilliant SYBR Green QPCR master mix (Stratagene) in a final volume of 25 l. Primer sequences were as follows: IE1 (sense, 5'-CAA GTG ACC GAG GAT TGC AA-3'; antisense, 5'-CAC CAT GTC CAC TCG AAC CTT-3'); IE2 (sense, 5'-TGA CCG AGG ATT GCA ACG A-3'; antisense, 5'-CGG CAT GAT TGA CAG CCT G-3').…”

Azasugar-Containing Phosphorothioate Oligonucleotide (AZPSON) DBM-2198 Inhibits Human Immunodeficiency Virus Type 1 (HIV-1) Replication by Blocking HIV-1 gp120 without Affecting the V3 Region

“…1 thioated substrate have been demonstrated to have antiviral activities by blocking viral entry (Luganini et al, 2008;Matsumura et al, 2009), replication (Torrence et al, 2006), packaging (Giannecchini et al, 2009;Takahashi et al, 2009), translation (Gonzalez-Carmona et al, 2013), and in some cases by targeting microRNA (Gebert et al, 2014;Lanford et al, 2010). Among P = S ONs studied as antiviral compounds, P = S ONs that inhibit HIV-1 infection have attracted attention.…”

“…These suggested mechanisms include blocking of viral adsorption (Luganini et al, 2008;Wyatt et al, 1994) and/or inhibition of HIV-1-specific enzymes, such as reverse transcriptase (Marshall and Caruthers, 1993;Marshall et al, 1992) or integrase (Jing and Hogan, 1998;Jing et al, 2000). P = S oligodeoxycytidine [poly (SdC) 28 ] interacts specifically with the positively-charged V3 loop of HIV-1 gp120 (Vaillant et al, 2006), possibly resulting in the inhibition of HIV-1 replication.…”