Murine cytomegalovirus major immediate-early protein 3 interacts with cellular and viral proteins in viral DNA replication compartments and is important for early gene activation

Murine cytomegalovirus (MCMV) immediate-early protein 3 (IE3) is essential for successful viral infection. This study developed MCMVs with an EGFP-fused IE3 gene in order to study IE3 gene expression, subnuclear distribution and biological function, as well as to examine the interaction of IE3 with cellular and viral proteins. The generated viruses included MCMVIE3gfp, in which IE1 was completely removed by the in-frame fusion of exons 3 and 5 and the C terminus of IE3 was tagged with EGFP, and MCMVIE1/3gfp, in which IE1 was kept intact and EGFP was also fused to the C terminus of IE3. Unlike human CMV (HCMV), whose growth was significantly reduced when IE2 (the HCMV homologue of IE3 in MCMV) was tagged with EGFP, MCMVs with IE3–EGFP presented an unchanged replication profile. Using these new constructs, the distribution of IE3 was revealed as well as its interaction with viral and cellular proteins, especially proteins pertaining to DNA replication (M44 and E1) and cellular intrinsic defence [promyelocytic leukemia protein and histone deacetylases (HDACs)]. It was also shown that IE3 domains co-localize with DNA replication domains, and IE3 attracted other required proteins into IE3 domains via protein–protein interactions. In addition, IE3 was shown to interact with HDAC2 and to eliminate the inhibitory effect of HDAC2 on early viral gene production. Together, these results suggest that IE3 acts as a key protein for viral DNA replication by establishing pre-replication domains via recruitment of the required viral and cellular proteins, and by reducing host defences

Syncytial and Congregative Effects of Dengue and Zika Viruses on the Aedes Albopictus Cell Line Differ among Viral Strains

Dengue viruses (DENV) and Zika virus (ZIKV) are transmitted among humans, or from non-human primates to humans, through mosquito bites. The interaction of the virus with mosquito cells is a key step in the viral life cycle. Therefore, the objective of this study was to determine how DENV and ZIKV interact with mosquito cells. Immunofluorescence assays and a direct visualization system were combined to monitor the syncytial or congregative effects of DENV and ZIKV strains on C6/36 cells. We examined the cytopathic effects of the strains on C6/36 mosquito cells, a widely used laboratory model for studying infection with these viruses. Our results indicated that all strains of DENV-1 and DENV-2, most DENV-4 strains, and some DENV-3 strains caused syncytial effects on C6/36 cells, whereas some DENV-3 and DENV-4 strains, and all tested ZIKV strains, caused cell congregation after infection but no cell fusion. In addition, we detected a range of environmental pH values from 6.0 to 8.0 supporting virus-induced cell fusion. The optimal pH condition was 7.5, at which viral production was also highest. Furthermore, the UV-inactivated virus did not cause cell fusion, thus suggesting that viral replication may be required for DENV’s syncytial effects on C6/36 cells. Syncytial and congregative effects of DENV and ZIKV on Aedes albopictus cells differ among viral strains. Syncytial effects of DENV on C6/36 are important for viral replication

Rapid Neutralization Testing System for Zika Virus Based on an Enzyme-Linked Immunospot Assay.

Zika virus (ZIKV) is a mosquito-borne flavivirus that has been associated with neuropathology in fetuses and adults, imposing a serious health concern. Therefore, the development of a vaccine is a global health priority. Notably, neutralization tests have a significant value for vaccine development and virus diagnosis. The cytopathic effect (CPE)-based neutralization test (Nt-CPE) is a common neutralization method for ZIKV. However, this method has some drawbacks, such as being time-consuming and labor-intensive and having low-throughput, which precludes its application in the detection of large numbers of specimens. To improve this problem, we developed a neutralization test based on an enzyme-linked immunospot assay (Nt-ELISPOT) for ZIKV and performed the assay in a 96-well format. A monoclonal antibody (mAb), 11C11, with high affinity and reactivity to ZIKV was used to detect ZIKV-infected cells. To optimize this method, the infectious dose of ZIKV was set at a multiplicity of infection (MOI) of 0.0625, and a detection experiment was performed after incubating for 24 h. As a result, under these conditions, the Nt-ELISPOT had good consistency with the traditional Nt-CPE to measure neutralizing titers of sera and neutralizing antibodies. Additionally, three neutralizing antibodies against ZIKV were screened by this method. Overall, we successfully developed an efficient neutralization test for ZIKV that is high-throughput and rapid. This Nt-ELISPOT can potentially be applied to detecting neutralizing titers of large numbers of specimens in vaccine evaluation and neutralizing antibody screening for ZIKV

Functional Interaction of Nuclear Domain 10 and Its Components with Cytomegalovirus after Infections: Cross-Species Host Cells versus Native Cells

Species-specificity is one of the major characteristics of cytomegaloviruses (CMVs) and is the primary reason for the lack of a mouse model for the direct infection of human CMV (HCMV). It has been determined that CMV cross-species infections are blocked at the post-entry level by intrinsic cellular defense mechanisms, but few details are known. It is important to explore how CMVs interact with the subnuclear structure of the cross-species host cell. In our present study, we discovered that nuclear domain 10 (ND10) of human cells was not disrupted by murine CMV (MCMV) and that the ND10 of mouse cells was not disrupted by HCMV, although the ND10-disrupting protein, immediate-early protein 1 (IE1), also colocalized with ND10 in cross-species infections. In addition, we found that the UL131-repaired HCMV strain AD169 (vDW215-BADrUL131) can infect mouse cells to produce immediate-early (IE) and early (E) proteins but that neither DNA replication nor viral particles were detectable in mouse cells. Unrepaired AD169 can express IE1 only in mouse cells. In both HCMV-infected mouse cells and MCMV-infected human cells, the knocking-down of ND10 components (PML, Daxx, and SP100) resulted in significantly increased viral-protein production. Our observations provide evidence to support our hypothesis that ND10 and ND10 components might be important defensive factors against the CMV cross-species infection

Single-cell multi-omics reveals dyssynchrony of the innate and adaptive immune system in progressive COVID-19.

Dysregulated immune responses against the SARS-CoV-2 virus are instrumental in severe COVID-19. However, the immune signatures associated with immunopathology are poorly understood. Here we use multi-omics single-cell analysis to probe the dynamic immune responses in hospitalized patients with stable or progressive course of COVID-19, explore V(D)J repertoires, and assess the cellular effects of tocilizumab. Coordinated profiling of gene expression and cell lineage protein markers shows that S100

One of the Triple Poly(A) Signals in the M112-113 Gene Is Important and Sufficient for Stabilizing the M112-113 mRNA and the Replication of Murine Cytomegalovirus

The M112-113 gene is the first early gene of the murine cytomegalovirus (MCMV), and its expression is activated by the immediate-early 3 (IE3) protein during MCMV infection in permissive cells. At its 5′ terminus, a 10-bp motif, upstream of the TATA box of the M112-113 gene, was identified to bind to IE3, and it is necessary for IE3 to activate M112-113 gene expression (Perez KJ et al. 2013 JVI). At the 3′ terminus of the M112-113 gene, three poly(A) signals (PASs) are arranged closely, forming a PAS cluster. We asked whether it is necessary to have the PAS cluster for the M112-113 gene and wondered which PAS is required or important for M112-113 gene expression. In this study, we mutated one, two, or all three PASs in expressing plasmids. Then, we applied bacterial artificial chromosome (BAC) techniques to mutate PASs in viruses. Gene expression and viral replication were analyzed. We found that not all three PASs are needed for M112-113 gene expression. Moreover, we revealed that just one of the three poly(A)s is enough for MCMV replication. However, the deletion of all three PASs did not kill MCMV, although it significantly attenuated viral replication. Finally, an mRNA stability assay was performed and demonstrated that PASs are important to stabilize M112-113 mRNA. Therefore, we conclude that just one of the PASs of the M112-113 gene is sufficient and important for MCMV replication through the stabilization of M112-113 mRNA

One of the Triple Poly(A) Signals in the M112-113 Gene Is Important and Sufficient for Stabilizing the M112-113 mRNA and the Replication of Murine Cytomegalovirus

The M112-113 gene is the first early gene of the murine cytomegalovirus (MCMV), and its expression is activated by the immediate-early 3 (IE3) protein during MCMV infection in permissive cells. At its 5′ terminus, a 10-bp motif, upstream of the TATA box of the M112-113 gene, was identified to bind to IE3, and it is necessary for IE3 to activate M112-113 gene expression (Perez KJ et al. 2013 JVI). At the 3′ terminus of the M112-113 gene, three poly(A) signals (PASs) are arranged closely, forming a PAS cluster. We asked whether it is necessary to have the PAS cluster for the M112-113 gene and wondered which PAS is required or important for M112-113 gene expression. In this study, we mutated one, two, or all three PASs in expressing plasmids. Then, we applied bacterial artificial chromosome (BAC) techniques to mutate PASs in viruses. Gene expression and viral replication were analyzed. We found that not all three PASs are needed for M112-113 gene expression. Moreover, we revealed that just one of the three poly(A)s is enough for MCMV replication. However, the deletion of all three PASs did not kill MCMV, although it significantly attenuated viral replication. Finally, an mRNA stability assay was performed and demonstrated that PASs are important to stabilize M112-113 mRNA. Therefore, we conclude that just one of the PASs of the M112-113 gene is sufficient and important for MCMV replication through the stabilization of M112-113 mRNA.</jats:p