Search

Purpose: To predict if developing human embryos are permissive to multiple coronaviruses., Method: We analyzed publicly available single-cell RNA-seq datasets of human embryos for the known canonical and non-canonical receptors and spike protein cleavage enzymes for multiple coronaviruses like SARS-CoV, SARS-CoV-2, MERS-CoV, hCoV-229E, and hCoV-NL63. We also analyzed the expression of host genes involved in viral replication, host proteins involved in viral endosomal sorting complexes required for transport (ESCRT), genes of host proteins that physically interact with proteins of SARS-CoV-2, and the host genes essential for coronavirus infectivity., Results: Of the known receptors of SARS viruses, ACE2, BSG, GOLGA7, and ZDHHC5 were expressed in different proportions in the zygote, 4-cell, 8-cell, morula, and blastocysts including the trophectoderm. The MERS-CoV receptor, DPP4, and hCoV-229E receptor, ANPEP, were expressed mainly from the compact morula to the blastocyst stages. Transcripts of the MERS-CoV alternate receptor LGALS1 were detected in most cells at all stages of development. TMPRSS2 transcripts were detected in the epiblast, primitive endoderm, and trophectoderm, while transcripts of the endosomal proteases CTSL, CTSB, and FURIN were expressed in most cells at all stages of development. ACE2 and TMPRSS2 were co-expressed in a proportion of epiblast and trophectoderm cells. The embryonic cells expressed genes involved in ESCRT, viral replication, SARS-CoV-2 interactions, and coronavirus infectivity. The ACE2 and TMPRSS2 co-expressing cells were enriched in genes associated with lipid metabolism, lysosome, peroxisome, and oxidative phosphorylation pathways., Conclusion: Preimplantation and implantation stage human embryos could be permissive to multiple hCoVs., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Promyelocytic leukemia protein (PML) is an essential organizer of PML nuclear bodies (NBs), which carry out a variety of activities, including antiviral functions. Herpesviruses from all subfamilies encode proteins that counteract PML NB-mediated antiviral defenses by multiple mechanisms. However, because of the species specificity of herpesviruses, only a limited number of in vivo studies have been undertaken to investigate the effect of PML or PML NBs on herpesvirus infection. To address this central issue in herpesvirus biology, we studied the course of infection in wild-type and PML⁻/⁻ mice using murine gammaherpesvirus 68 (MHV68), which encodes a tegument protein that induces PML degradation. While acute infection in PML⁻/⁻ mice progressed similarly to that in wild-type mice, the lytic reactivation frequency was higher in peritoneal exudate cells, due to both an increase of MHV68 genome-positive cells and greater reactivation efficiency. We also detected a higher frequency of persistent infection in PML⁻/⁻ peritoneal cells. These findings suggest that the PML protein can repress the establishment or maintenance of gammaherpesvirus latency in vivo. Further use of the PML⁻/⁻ mouse model should aid in dissecting the molecular mechanisms that underlie the role of PML in gammaherpesvirus latency and may yield clues for how PML modulates herpesvirus latency in general.

Intracellular protein trafficking pathways are hijacked by viruses at various stages of viral life-cycle. Heterotetrameric adaptor protein complexes (APs) mediate vesicular trafficking at distinct intracellular sites and are essential for maintaining the organellar homeostasis. In the present study, we studied the effect of AP-1 and AP-3 deficiency on flavivirus infection in cells functionally lacking these proteins. We show that AP-1 and AP-3 participate in flavivirus life-cycle at distinct stages. AP-3-deficient cells showed delay in initiation of Japanese encephalitis virus and dengue virus RNA replication, which resulted in reduction of infectious virus production. AP-3 was found to colocalize with RNA replication compartments in infected wild-type cells. AP-1 deficiency affected later stages of dengue virus infection where increased intracellular accumulation of infectious virus was observed. Therefore, our results propose a novel role for AP-1 and AP-3 at distinct stages of infection of some of the RNA viruses.

The largest isoform of adenovirus early region 1A (E1A) contains a unique region termed conserved region 3 (CR3). This region activates viral gene expression by recruiting cellular transcription machinery to the early viral promoters. Recent studies have suggested that there is an optimal level of E1A-dependent transactivation required by human adenovirus (hAd) during infection and that this may be achieved via functional cross talk between the N termini of E1A and CR3. The N terminus of E1A binds GCN5, a cellular lysine acetyltransferase (KAT). We have identified a second independent interaction of E1A with GCN5 that is mediated by CR3, which requires residues 178 to 188 in hAd5 E1A. GCN5 was recruited to the viral genome during infection in an E1A-dependent manner, and this required both GCN5 interaction sites on E1A. Ectopic expression of GCN5 repressed transactivation by both E1A CR3 and full-length E1A. In contrast, RNA interference (RNAi) depletion of GCN5 or treatment with the KAT inhibitor cyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl]hydrazone (CPTH2) resulted in increased E1A CR3 transactivation. Moreover, activation of the adenovirus E4 promoter by E1A was increased during infection of homozygous GCN5 KAT-defective (hat/hat) mouse embryonic fibroblasts (MEFs) compared to wild-type control MEFs. Enhanced histone H3 K9/K14 acetylation at the viral E4 promoter required the newly identified binding site for GCN5 within CR3 and correlated with repression and reduced occupancy by phosphorylated RNA polymerase II. Treatment with CPTH2 during infection also reduced virus yield. These data identify GCN5 as a new negative regulator of transactivation by E1A and suggest that its KAT activity is required for optimal virus replication.

Human interferon (IFN)-inducible IFI16 protein, an innate immune sensor of intracellular DNA, modulates various cell functions, however, its role in regulating virus growth remains unresolved. Here, we adopt two approaches to investigate whether IFI16 exerts pro- and/or anti-viral actions. First, the IFI16 gene was silenced using specific small interfering RNAs (siRNA) in human embryo lung fibroblasts (HELF) and replication of DNA and RNA viruses evaluated. IFI16-knockdown resulted in enhanced replication of Herpesviruses, in particular, Human Cytomegalovirus (HCMV). Consistent with this, HELF transduction with a dominant negative form of IFI16 lacking the PYRIN domain (PYD) enhanced the replication of HCMV. Second, HCMV replication was compared between HELFs overexpressing either the IFI16 gene or the LacZ gene. IFI16 overexpression decreased both virus yield and viral DNA copy number. Early and late, but not immediate-early, mRNAs and proteins were strongly down-regulated, thus IFI16 may exert its antiviral effect by impairing viral DNA synthesis. Constructs with the luciferase reporter gene driven by deleted or site-specific mutated forms of the HCMV DNA polymerase (UL54) promoter demonstrated that the inverted repeat element 1 (IR-1), located between -54 and -43 relative to the transcription start site, is the target of IFI16 suppression. Indeed, electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated that suppression of the UL54 promoter is mediated by IFI16-induced blocking of Sp1-like factors. Consistent with these results, deletion of the putative Sp1 responsive element from the HCMV UL44 promoter also relieved IFI16 suppression. Together, these data implicate IFI16 as a novel restriction factor against HCMV replication and provide new insight into the physiological functions of the IFN-inducible gene IFI16 as a viral restriction factor.

The ORF49 gene product (ORF49p) of the varicella-zoster virus (VZV) is likely a myristylated tegument protein, and its homologs are conserved across the herpesvirus subfamilies. The UL11 gene of herpes simplex virus type 1 and of pseudorabies virus and the UL99 gene of human cytomegalovirus are the homologs of ORF49 and have been well characterized by using mutant viruses; however, little research on the VZV ORF49 gene has been reported. Here we report on VZV ORF49p expression, subcellular localization, and effect on viral spread in vitro. ORF49p was expressed during the late phase of infection and located in the juxtanuclear region of the cytoplasm, where it colocalized mainly with the trans-Golgi network-associated protein. ORF49p was incorporated into virions and showed a molecular mass of 13 kDa in VZV-infected cells and virions. To elucidate the role of the ORF49 gene, we constructed a mutant virus that lacked a functional ORF49. No differences in plaque size or cell-cell spread were observed in human embryonic fibroblast cells, MRC-5 cells, infected with the wild-type or the mutant virus. However, the mutant virus showed diminished cell-cell infection in a human malignant melanoma cell line, MeWo cells. Therefore, VZV ORF49p is important for virus growth in MeWo cells, but not in MRC-5 cells. VZV may use different mechanisms for virus growth in MeWo and MRC-5 cells. If so, understanding the role of ORF49p should help elucidate how VZV accomplishes cell-cell infections in different cell types.

Dengue virus, a member of the family Flaviviridae, poses a serious public health threat worldwide. Dengue virus is a positive-sense RNA virus that harbors a genome of approximately 10.7 kb. Replication of dengue virus is mediated coordinately by cis-acting genomic sequences, viral proteins, and host cell factors. We have isolated and identified several host cell factors from baby hamster kidney cell extracts that bind with high specificity and high affinity to sequences within the untranslated regions of the dengue virus genome. Among the factors identified, Y box-binding protein-1 (YB-1) and the heterogeneous nuclear ribonucleoproteins (hnRNPs), hnRNP A1, hnRNP A2/B1, and hnRNP Q, bind to the dengue virus 3'-untranslated region. Further analysis indicated that YB-1 binds to the dengue virus 3' stem loop, a conserved structural feature located at the 3' terminus of the 3'-untranslated region of many flaviviruses. Analysis of the impact of YB-1 on replication of dengue virus in YB-1+/+ and YB-1-/- mouse embryo fibroblasts indicated that host YB-1 mediates an antiviral effect. Further studies demonstrated that this antiviral impact is due, at least in part, to a repressive role of YB-1 on dengue virus translation via a mechanism that requires viral genomic sequences. These results suggest a novel role for YB-1 as an antiviral host cell factor.

This research evaluated the ability of phosphonoformic acid to inhibit bovine herpesvirus 1 (BHV-1) in cumulus cells commonly used in co-culture with bovine in vitro-produced embryos. At 200 and 400 microg/ml, phosphonoformic acid inhibited 4 logs of BHV-1. Subsequently, phosphonoformic acid (200 and 400 microg/ml) added to both in vitro fertilization and culture medium resulted in a decrease in the proportion of developed blastocysts, and the number of cells per blastocyst was lower in the treated embryos. Therefore, while phosphonoformic acid can effectively inhibit replication of BHV-1 in co-culture cells, it also inhibits development of in vitro-produced bovine embryos.

Recent studies have shown that exposed, in vitro-derived embryos remain contaminated with bovine viral diarrhea virus (BVDV) after washing. However, introduction of a Genotype II versus Genotype I strain of BVDV into an IVF system was reported to provide greater potential for transmission of disease. The primary objective of this study was to compare the potentials for different strains of noncytopathic BVDV to replicate in an IVF system, associate with IVF embryos and infect co-cultured cells via association with washed embryos. The secondary objective was to compare the effect of different strains of BVDV on embryonic development. Two Genotype I (SD-1 and NY-1) and 2 Genotype II (CD-87 and PA-131) strains of BVDV were evaluated. After IVM and IVF of oocytes, presumptive zygotes were washed and transferred into in vitro cultures containing uterine tubal cells (UTC) and medium that was free of BVDV-neutralizing activity. Immediately before addition of zygotes, the cultures were inoculated with 10(5) cell culture infective doses (50%, CCID50) of a strain of BVDV or maintained as a negative control. Cultures of zygotes were then incubated for 7 d. Embryonic development was observed on Days 3 and 7, and attempts were made to isolate BVDV from UTC and medium on Day 7. Also on Day 7, groups of intact, washed blastocysts were either transferred into virus-free secondary cultures containing UTC or sonicated with sonicate fluid assayed by both virus isolation and single-closed-tube reverse transcription nested polymerase chain reaction (RT-nPCR). After 3 d in secondary culture, hatched embryos were enumerated, and medium from the cultures, washed UTC and embryos were tested for BVDV by virus isolation. In addition, washed UTC and embryos were tested for BVDV using RT-nPCR. All strains of BVDV persisted and replicated in the embryo culture environment, but cleavage beyond the 4-cell stage, blastocyst development and hatching varied among cultures contaminated with different strains of virus. Further, the quantity of BVDV associated with washed embryos from both initial and secondary cultures varied among strains, but the variation was unrelated to difference in genotype (SD-1 and PA-131 greater than NY-1 and CD-87). Although all strains of BVDV replicated in UTC in the initial in vitro cultures and remained associated with washed blastocysts, susceptible UTC in the secondary in vitro cultures were seldom infected by any strain of virus.

The aim of the present study was to determine whether or not cytopathic (CP) and noncytopathic (NCP) bovine viral diarrhea virus (BVDV) are able to replicate within in vitro-produced embryos and to investigate whether inoculation of embryos with BVDV affects their normal development. Zona pellucida (ZP)-free oocytes, zygotes, 8-cell-stage embryos, morulae, and hatched blastocysts (HB) were incubated for 1 h in 1 ml of Minimal Essential Medium containing 10(6.00) tissue culture infectious dose (TCID)50/ml NCP BVDV isolate 22,146 or 10(6.25) TCID50/ml CP BVDV strain Oregon C24V. At 0, 12, 24, 36, 48, 60, and 72 h postinoculation (hpi), groups of embryos were collected for virus titration. A small amount of newly produced virus was detected in 8-cell embryos at 60 hpi (10(1.8) TCID50/100 cells), but only for CP BVDV. For ZP-free morulae and HB, maximal intracellular virus titers were, respectively, 10(1.47) and 10(2.33) TCID50/100 cells at 48 hpi for the CP biotype and 10(0.64) and 10(0.84) TCID50/100 cells at 72 hpi for the NCP biotype. Only an infection with CP BVDV had a significant inhibitory effect on further development of ZP-free morulae. It can be concluded that ZP-free in vitro-produced embryos are permissive to an infection with BVDV, with increasing susceptibility of the embryos in accordance with their developmental stage. In contrast to observations in ZP-free in vitro-produced embryos, no virus replication or signs of embryonic degeneration were detected in ZP-intact in vitro-derived embryos.

Replication of murine coronaviruses in eight mouse embryonic stem (ES) cell lines of several genetic backgrounds was examined. Both mouse hepatitis virus (MHV) type 2 and MHV, strain A59 replicated well with no or minimal cytopathic effect in all the ES cell lines tested. The results suggest the possibility that MHV-infected ES cells may disseminate MHV in mouse colonies due to embryo manipulation.