Recombinant Lloviu virus as a tool to study viral replication and host responses

Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness.     

Lytic viral replication and immunopathology in a cytomegalovirus-induced mouse model of secondary hemophagocytic lymphohistiocytosis

Background

Hemophagocytic lymphohistiocytosis (HLH) is a rare immunological disorder caused by unbridled activation of T cells and macrophages, culminating in a life-threatening cytokine storm. A genetic and acquired subtype are distinguished, termed primary and secondary HLH, respectively. Clinical manifestations of both forms are frequently preceded by a viral infection, predominantly with herpesviruses. The exact role of the viral infection in the development of the hemophagocytic syndrome remains to be further elucidated.

Methods

We utilized a recently developed murine model of cytomegalovirus-associated secondary HLH and dissected the respective contributions of lytic viral replication and immunopathology in its pathogenesis.

Results

HLH-like disease only developed in cytomegalovirus-susceptible mouse strains unable to clear the virus, but the severity of symptoms was not correlated to the infectious viral titer. Lytic viral replication and sustained viremia played an essential part in the pathogenesis since abortive viral infection was insufficient to induce a full-blown HLH-like syndrome. Nonetheless, a limited set of symptoms, in particular anemia, thrombocytopenia and elevated levels of soluble CD25, appeared less dependent of the viral replication but rather mediated by the host’s immune response, as corroborated by immunosuppressive treatment of infected mice with dexamethasone.

Conclusion

Both virus-mediated pathology and immunopathology cooperate in the pathogenesis of full-blown virus-associated secondary HLH and are closely entangled. A certain level of viremia appears necessary to elicit the characteristic HLH-like symptoms in the model.

     

Epstein-Barr virus infection and sporadic breast cancer risk: a meta-analysis

Background

A large number of epidemiological studies have evaluated the association between Epstein-Barr virus infection and breast carcinoma risk but results have been inconsistent.

Methodology

Research using the polymerase chain reaction technique for detecting the Epstein-Barr virus was selected; 24 studies and 1535 cases were reviewed. Information on the study populations, sample types, publication calendar period and histological types of breast carcinoma were collected. An unconditional logistic regression model was used to analyze potential parameters related to the Epstein-Barr virus prevalence. A Kappa test was used to evaluate the consistency in detecting different Epstein-Barr virus DNA regions. Nine studies that included control groups and 1045 breast cancer cases were adopted in this meta-analysis.

Conclusions

We found that 29.32% of the patients with breast carcinoma were infected with the Epstein-Barr virus. The prevalence of Epstein-Barr was highest in Asia (35.25%) and lowest in the USA (18.27%). Statistical analysis revealed a trend that showed lobular breast carcinoma might have the strongest association with Epstein-Barr virus infection. This meta-analysis showed a significant increase in breast malignancy risk in patients testing positive for the Epstein-Barr virus (OR = 6.29, 95% CI = 2.13–18.59). This result suggests that an Epstein-Barr virus infection is statistically associated with increased breast carcinoma risk.     

Tetrameric ring formation of Epstein-Barr virus polymerase processivity factor is crucial for viral replication

The Epstein-Barr virus BMRF1 DNA polymerase processivity factor, which is essential for viral genome replication, exists mainly as a C-shaped head-to-head homodimer but partly forms a ring-shaped tetramer through tail-to-tail association. Based on its molecular structure, several BMRF1 mutant viruses were constructed to examine their influence on viral replication. The R256E virus, which has a severely impaired capacity for DNA binding and polymerase processivity, failed to form replication compartments, resulting in interference of viral replication, while the C95E mutation, which impairs head-to-head contact in vitro, unexpectedly hardly affected the viral replication. Also, surprisingly, replication of the C206E virus, which is expected to have impairment of tail-to-tail contact, was severely restricted, although the mutant protein possesses the same in vitro biochemical activities as the wild type. Since the tail-to-tail contact surface is smaller than that of the head-to-head contact area, its contribution to ring formation might be essential for viral replication.     

Epstein-Barr virus stimulates torque teno virus replication: a possible relationship to multiple sclerosis

Viral infections have been implicated in the pathogenesis of multiple sclerosis. Epstein-Barr virus (EBV) has frequently been investigated as a possible candidate and torque teno virus (TTV) has also been discussed in this context. Nevertheless, mechanistic aspects remain unresolved. We report viral replication, as measured by genome amplification, as well as quantitative PCR of two TTV-HD14 isolates isolated from multiple sclerosis brain in a series of EBV-positive and -negative lymphoblastoid and Burkitt's lymphoma cell lines. Our results demonstrate the replication of both transfected TTV genomes up to day 21 post transfection in all the evaluated cell lines. Quantitative amplification indicates statistically significant enhanced TTV replication in the EBV-positive cell lines, including the EBV-converted BJAB line, in comparison to the EBV-negative Burkitt's lymphoma cell line BJAB. This suggests a helper effect of EBV infections in the replication of TTV. The present study provides information on a possible interaction of EBV and TTV in the etiology and progression of multiple sclerosis.     

Architecture of replication compartments formed during Epstein-Barr virus lytic replication

Epstein-Barr virus (EBV) productive DNA replication occurs at discrete sites, called replication compartments, in nuclei. In this study we performed comprehensive analyses of the architecture of the replication compartments. The BZLF1 oriLyt binding proteins showed a fine, diffuse pattern of distribution throughout the nuclei at immediate-early stages of induction and then became associated with the replicating EBV genome in the replication compartments during lytic infection. The BMRF1 polymerase (Pol) processivity factor showed a homogenous, not dot-like, distribution in the replication compartments, which completely coincided with the newly synthesized viral DNA. Inhibition of viral DNA replication with phosphonoacetic acid, a viral DNA Pol inhibitor, eliminated the DNA-bound form of the BMRF1 protein, although the protein was sufficiently expressed in the cells. These observations together with the findings that almost all abundantly expressed BMRF1 proteins existed in the DNA-bound form suggest that the BMRF1 proteins not only act at viral replication forks as Pol processive factors but also widely distribute on newly replicated EBV genomic DNA. In contrast, the BALF5 Pol catalytic protein, the BALF2 single-stranded-DNA binding protein, and the BBLF2/3 protein, a component of the helicase-primase complex, were colocalized as distinct dots distributed within replication compartments, representing viral replication factories. Whereas cellular replication factories are constructed based on nonchromatin nuclear structures and nuclear matrix, viral replication factories were easily solubilized by DNase I treatment. Thus, compared with cellular DNA replication, EBV lytic DNA replication factories would be simpler so that construction of the replication domain would be more relaxed.     

Deletion of Epstein-Barr virus BFLF2 leads to impaired viral DNA packaging and primary egress as well as to the production of defective viral particles

Previous genetic and biochemical studies performed with several members of the Alphaherpesvirus subfamily have shown that the UL31 and UL34 proteins are essential components of the molecular machinery that mediates the primary egress of newly assembled capsids across the nuclear membrane. Further, there is substantial evidence that BFLF2 and BFRF1, the respective positional homologs of UL31 and UL34 in the Epstein-Barr virus (EBV) genome, are also their functional homologs, i.e., that the UL31/UL34 pathway is common to distant herpesviruses. However, the low degree of protein sequence identity between UL31 and BFLF2 would argue against such a hypothesis. To further clarify this issue, we have constructed a recombinant EBV strain devoid of BFLF2 (DeltaBFLF2) and show that BFLF2 is crucial for efficient virus production but not for lytic DNA replication or B-cell transformation. This defective phenotype could be efficiently restored by trans complementation with a BFLF2 expression plasmid. Detailed analysis of replicating cells by electron microscopy revealed that, as expected, DeltaBFLF2 viruses not only failed to egress from the nucleus but also showed defective DNA packaging. Nonfunctional primary egress did not, however, impair the production and extracellular release of enveloped but empty viral particles that comprised L particles containing tegument-like structures and a few virus-like particles carrying empty capsids. The DeltaBFLF2 and DeltaUL31 phenotypes therefore only partly overlap, from which we infer that BFLF2 and UL31 have substantially diverged during evolution to fulfil related but distinct functions.     

Plasticity of DNA replication initiation in Epstein-Barr virus episomes

In mammalian cells, the activity of the sites of initiation of DNA replication appears to be influenced epigenetically, but this regulation is not fully understood. Most studies of DNA replication have focused on the activity of individual initiation sites, making it difficult to evaluate the impact of changes in initiation activity on the replication of entire genomic loci. Here, we used single molecule analysis of replicated DNA (SMARD) to study the latent duplication of Epstein-Barr virus (EBV) episomes in human cell lines. We found that initiation sites are present throughout the EBV genome and that their utilization is not conserved in different EBV strains. In addition, SMARD shows that modifications in the utilization of multiple initiation sites occur across large genomic regions (tens of kilobases in size). These observations indicate that individual initiation sites play a limited role in determining the replication dynamics of the EBV genome. Long-range mechanisms and the genomic context appear to play much more important roles, affecting the frequency of utilization and the order of activation of multiple initiation sites. Finally, these results confirm that initiation sites are extremely redundant elements of the EBV genome. We propose that these conclusions also apply to mammalian chromosomes.     

Interaction with the Epstein-Barr virus helicase targets Zta to DNA replication compartments

Zta has a dual role in the Epstein-Barr virus (EBV) lytic cycle, acting as a key regulator of EBV lytic gene expression and also being essential for lytic viral DNA replication. Zta's replication function is mediated in part through interactions with the core viral replication proteins. We now show interaction between Zta and the helicase (BBLF4) and map the binding region to within amino acids (aa) 22 to 86 of the Zta activation domain. In immunofluorescence assays, green fluorescent protein (GFP)-tagged BBLF4 localized to the cytoplasm of transfected cells. Cotransfection of Zta resulted in translocation of BBLF4-GFP into the nucleus indicating interaction between these two proteins. However, Zta with a deletion of aa 24 to 86 was unable to mediate nuclear translocation of BBLF4-GFP. Results obtained with Zta variants carrying deletions across the aa 24 to 86 region indicated more than one contact site for BBLF4 within this domain, and this was reinforced by the behavior of the four-point mutant Zta (m22/26,74/75), which was severely impaired for BBLF4 interaction. Binding of BBLF4 to Zta was confirmed using GST affinity assays. In both cotransfection-replication assays and replication assays performed in EBV-positive P3HR1 cells, the Zta (m22/26,74/75) mutant was replication defective. In Zta-transfected D98-HR1 cells, replication compartments could be detected by immunofluorescence staining using anti-BMRF1 monoclonal antibody. Cells transfected with Zta variants that were defective for helicase binding still formed replication compartments, but Zta was excluded from these compartments. These experiments reveal a role for the Zta-helicase interaction in targeting Zta to sites of viral DNA replication.     

Modification of Epstein-Barr virus replication by tunicamycin.

The effect of tunicamycin, which inhibits N-linked glycosylation, on the replication of Epstein-Barr virus was examined. Tunicamycin markedly reduced the yield of virus from producing cells. At concentrations of 1 to 2 micrograms of tunicamycin per ml, there was a buildup of intracellular virus in P3HR1-Cl13 cells but not in MCUV5 cells; at a concentration of 5 micrograms of tunicamycin per ml in P3HR1-Cl13 cells, viral DNA synthesis was inhibited as well. Viral glycoproteins lacking N-linked sugars were apparently inserted into the cell membrane, and the small amount of virus made in the presence of drug was able to bind specifically to its receptor on B cells. However, the ability of the virus to induce immunoglobulin secretion by fresh human lymphocytes was impaired. This implies a role for viral glycoproteins in the penetration as well as the attachment of virus.     

Postreplicative mismatch repair factors are recruited to Epstein-Barr virus replication compartments

The mismatch repair (MMR) system, highly conserved throughout evolution, corrects nucleotide mispairing that arise during cellular DNA replication. We report here that proliferating cell nuclear antigen (PCNA), the clamp loader complex (RF-C), and a series of MMR proteins like MSH-2, MSH-6, MLH1, and hPSM2 can be assembled to Epstein-Barr virus replication compartments, the sites of viral DNA synthesis. Levels of the DNA-bound form of PCNA increased with progression of viral productive replication. Bromodeoxyuridine-labeled chromatin immunodepletion analyses confirmed that PCNA is loaded onto newly synthesized viral DNA as well as BALF2 and BMRF1 viral proteins during lytic replication. Furthermore, the anti-PCNA, -MSH2, -MSH3, or -MSH6 antibodies could immunoprecipitate BMRF1 replication protein probably via the viral DNA genome. PCNA loading might trigger transfer of a series of host MMR proteins to the sites of viral DNA synthesis. The MMR factors might function for the repair of mismatches that arise during viral replication or act to inhibit recombination between moderately divergent (homologous) sequences.