Impact of Amino Acid Substitutions in Region II and Helix K of Herpes Simplex Virus 1 and Human Cytomegalovirus DNA Polymerases on Resistance to Foscarnet.

Amino acid substitutions conferring resistance of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) to foscarnet (PFA) are located in the genes UL30 and UL54, respectively, encoding the DNA polymerase (pol). In this study, we analyzed the impact of substitutions located in helix K and region II that are involved in the conformational changes of the DNA pol. Theoretical substitutions were identified by sequences alignment of the helix K and region II of human herpesviruses (susceptible to PFA) and bacteriophages (resistant to PFA) and introduced in viral genomes by recombinant phenotyping. We characterized the susceptibility of HSV-1 and HCMV mutants to PFA. In UL30, the substitutions I619K (helix K), V715S, and A719T (both in region II) increased mean PFA 50% effective concentrations (EC50s) by 2.5-, 5.6-, and 2.0-fold, respectively, compared to the wild type (WT). In UL54, the substitution Q579I (helix K) conferred hypersusceptibility to PFA (0.17-fold change), whereas the substitutions Q697P, V715S, and A719T (all in region II) increased mean PFA EC50s by 3.8-, 2.8- and 2.5-fold, respectively, compared to the WT. These results were confirmed by enzymatic assays using recombinant DNA pol harboring these substitutions. Three-dimensional modeling suggests that substitutions conferring resistance/hypersusceptibility to PFA located in helix K and region II of UL30 and UL54 DNA pol favor an open/closed conformation of these enzymes, resulting in a lower/higher drug affinity for the proteins. Thus, this study shows that both regions of UL30 and UL54 DNA pol are involved in the conformational changes of these proteins and can influence the susceptibility of both viruses to PFA.

Differential impact of various substitutions at codon 715 in region II of HSV-1 and HCMV DNA polymerases.

This study aimed at understanding the impact of different substitutions at codon 715 localized in the region II of the palm domain of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) DNA polymerases (pol). Here, we report a new theoretical mutation V715S that confers resistance of HSV-1 to foscarnet/acyclovir (5.6- and 9.2-fold increases EC50 values compared to wild type, respectively) and of HCMV to foscarnet/ganciclovir (2.8- and 2.9-fold increases in EC50 values compared to wild type, respectively). To further analyze the importance of this amino acid, we investigated the impact of the already known mutations V715M and V715G on the replicative capacities and drug susceptibilities of both viruses as well as on the activity and drug inhibition of the DNA pol. The V715G recombinant HSV-1 mutant was resistant to foscarnet and acyclovir (3.4- and 4.6-fold EC50 increase, respectively) whereas the V715M mutant was susceptible to foscarnet and resistant to acyclovir (3.4-fold EC50 increase). The V715G recombinant HCMV mutant did not grow and the V715M mutant was resistant to foscarnet (3.7-fold EC50 increase) and susceptible to ganciclovir. Finally, we showed by three-dimensional modeling that the differential impact of these mutations on the viral replicative capacity and drug resistance profile was related to different hydrophobic local environments for V715 in the DNA pol of the two viruses. Furthermore, we hypothesize that the DNA pol of HSV-1 is more tolerant to changes at this residue compared to that of HCMV because of a more hydrophobic environment stabilizing the region.

An Early Microglial Response Is Needed To Efficiently Control Herpes Simplex Virus Encephalitis.

The role of a signaling pathway through macrophage colony-stimulating factor (MCSF) and its receptor, macrophage colony-stimulating factor 1 receptor (CSF1R), during experimental herpes simplex virus 1 (HSV-1) encephalitis (HSE) was studied by two different approaches. First, we evaluated the effect of stimulation of the MCSF/CSF1R axis before infection. Exogenous MCSF (40 µg/kg of body weight intraperitoneally [i.p.]) was administered once daily to BALB/c mice on days 4 and 2 before intranasal infection with 2,500 PFU of HSV-1. MCSF treatment significantly increased mouse survival compared to saline (50% versus 10%; P = 0.0169). On day 6 postinfection (p.i.), brain viral titers were significantly decreased, whereas beta interferon (IFN-ß) was significantly increased in mice treated with MCSF compared to mice treated with saline. The number of CD68+ (a phagocytosis marker) microglial cells was significantly increased in MCSF-treated mice compared to the saline-treated group. Secondly, we conditionally depleted CSF1R on microglial cells of CSF1R-loxP-CX3CR1-cre/ERT2 mice (in a C57BL/6 background) through induction with tamoxifen. The mice were then infected intranasally with 600,000 PFU of HSV-1. The survival rate of mice depleted of CSF1R (knockout [KO] mice) was significantly lower than that of wild-type (WT) mice (0% versus 67%). Brain viral titers and cytokine/chemokine levels were significantly higher in KO than in WT animals on day 6 p.i. Furthermore, increased infiltration of monocytes into the brains of WT mice was seen on day 6 p.i., but not in KO mice. Our results suggest that microglial cells are essential to control HSE at early stages of the disease and that the MCSF/CSF1R axis could be a therapeutic target to regulate their response to infection.IMPORTANCE Microglia appear to be one of the principal regulators of neuroinflammation in the central nervous system (CNS). An increasing number of studies have demonstrated that the activation of microglia could result in either beneficial or detrimental effects in different CNS disorders. Hence, the role of microglia during herpes simplex virus encephalitis (HSE) has not been fully characterized. Using experimental mouse models, we showed that an early activation of the MCSF/CSF1R axis improved the outcome of the disease, possibly by inducing a proliferation of microglia. In contrast, depletion of microglia before HSV-1 infection worsened the prognosis of HSE. Thus, an early microglial response followed by sustained infiltration of monocytes and T cells into the brain seem to be key components for a better clinical outcome. These data suggest that microglia could be a potential target for immunomodulatory strategies combined with antiviral therapy to better control the outcome of this devastating disease.

Hypersusceptibility of Human Cytomegalovirus to Foscarnet Induced by Mutations in Helices K and P of the Viral DNA Polymerase.

Herein, we phenotypically and enzymatically characterize the theoretical mutation Q579I in helix K and the already described clinical mutation K805Q in helix P of cytomegalovirus DNA polymerase for susceptibility to foscarnet. Q579I and K805Q recombinant viruses were hypersusceptible to foscarnet (respective mean 50% effective concentrations [EC50] of 0.12- and 0.19-fold that of the wild type). Three-dimensional modeling analysis suggested that both mutations favor the closed conformation of the enzyme to which foscarnet binds with a higher affinity.

Herpesvirus DNA polymerases: Structures, functions and inhibitors.

Human herpesviruses are large double-stranded DNA viruses belonging to the Herpesviridae family. These viruses have the ability to establish lifelong latency into the host and to periodically reactivate. Primary infections and reactivations of herpesviruses cause a large spectrum of diseases and may lead to severe complications in immunocompromised patients. The viral DNA polymerase is a key enzyme in the lytic phase of the infection by herpesviruses. This review focuses on the structures and functions of viral DNA polymerases of herpes simplex virus (HSV) and human cytomegalovirus (HCMV). DNA polymerases of HSV (UL30) and HCMV (UL54) belong to B family DNA polymerases with which they share seven regions of homology numbered I to VII as well as a δ-region C which is homologous to DNA polymerases δ. These DNA polymerases are multi-functional enzymes exhibiting polymerase, 3'-5' exonuclease proofreading and ribonuclease H activities. Furthermore, UL30 and UL54 DNA polymerases form a complex with UL42 and UL44 processivity factors, respectively. The mechanisms involved in their polymerisation activity have been elucidated based on structural analyses of the DNA polymerase of bacteriophage RB69 crystallized under different conformations, i.e. the enzyme alone or in complex with DNA and with both DNA and incoming nucleotide. All antiviral agents currently used for the prevention or treatment of HSV and HCMV infections target the viral DNA polymerases. However, long-term administration of these antivirals may lead to the emergence of drug-resistant isolates harboring mutations in genes encoding viral enzymes that phosphorylate drugs (i.e., nucleoside analogues) and/or DNA polymerases.

MxA Mediates SUMO-Induced Resistance to Vesicular Stomatitis Virus.

UNLABELLED: Multiple cellular pathways are regulated by small ubiquitin-like modifier (SUMO) modification, including ubiquitin-mediated proteolysis, signal transduction, innate immunity, and antiviral defense. In the study described in this report, we investigated the effects of SUMO on the replication of two members of the Rhabdoviridae family, vesicular stomatitis virus (VSV) and rabies virus (RABV). We show that stable expression of SUMO in human cells confers resistance to VSV infection in an interferon-independent manner. We demonstrate that SUMO expression did not alter VSV entry but blocked primary mRNA synthesis, leading to a reduction of viral protein synthesis and viral production, thus protecting cells from VSV-induced cell lysis. MxA is known to inhibit VSV primary transcription. Interestingly, we found that the MxA protein was highly stabilized in SUMO-expressing cells. Furthermore, extracts from cells stably expressing SUMO exhibited an increase in MxA oligomers, suggesting that SUMO plays a role in protecting MxA from degradation, thus providing a stable intracellular pool of MxA available to combat invading viruses. Importantly, MxA depletion in SUMO-expressing cells abrogated the anti-VSV effect of SUMO. Furthermore, SUMO expression resulted in interferon-regulatory factor 3 (IRF3) SUMOylation, subsequently decreasing RABV-induced IRF3 phosphorylation and interferon synthesis. As expected, this rendered SUMO-expressing cells more sensitive to RABV infection, even though MxA was stabilized in SUMO-expressing cells, since its expression did not confer resistance to RABV. Our findings demonstrate opposing effects of SUMO expression on two viruses of the same family, intrinsically inhibiting VSV infection through MxA stabilization while enhancing RABV infection by decreasing IFN induction. IMPORTANCE: We report that SUMO expression reduces interferon synthesis upon RABV or VSV infection. Therefore, SUMO renders cells more sensitive to RABV but unexpectedly renders cells resistant to VSV by blocking primary mRNA synthesis. Unlike the interferon-mediated innate immune response, intrinsic antiviral resistance is mediated by constitutively expressed restriction factors. Among the various anti-VSV restriction factors, only MxA is known to inhibit VSV primary transcription, and we show here that its expression does not alter RABV infection. Interestingly, MxA depletion abolished the inhibition of VSV by SUMO, demonstrating that MxA mediates SUMO-induced intrinsic VSV resistance. Furthermore, MxA oligomerization is known to be critical for its protein stability, and we show that higher levels of oligomers were formed in cells expressing SUMO than in wild-type cells, suggesting that SUMO may play a role in protecting MxA from degradation, providing a stable intracellular pool of MxA able to protect cells from viral infection.