Adenoviral vectors infect B lymphocytes in vivo.

B cells are the antibody-producing arm of the adaptive immune system and play a critical role in controlling pathogens. Several groups have now demonstrated the feasibility of using engineered B cells as a therapy, including infectious disease control and gene therapy of serum deficiencies. These studies have largely utilized ex vivo modification of the cells. Direct in vivo engineering would be of utility to the field, particularly in infectious disease control where the infrastructure needs of ex vivo cell modification would make a broad vaccination campaign highly challenging. In this study we demonstrate that engineered adenoviral vectors are capable of efficiently transducing murine and human primary B cells both ex vivo and in vivo. We found that unmodified human adenovirus C5 was capable of infecting B cells in vivo, likely due to interactions between the virus penton base protein and integrins. We further describe vector modification with B cell-specific gene promoters and successfully restrict transgene expression to B cells, resulting in a strong reduction in gene expression from the liver, the main site of human adenovirus C5 infection in vivo.

Heterogeneous Nuclear Ribonucleoproteins Participate in Hepatitis E Virus Replication.

Coordinated assembly of viral and host factors is essential for the successful propagation of viruses as well as the generation of host antiviral response. Previous studies from our group, as well as from other groups, have identified host proteins interacting with various components of the hepatitis E virus (HEV). However, the functional relevance of host protein interactions in HEV replication context has been notably overlooked. The present study reports that heterogeneous nuclear ribonucleoproteins (hnRNPs), namely hnRNPK, hnRNPA2B1, hnRNPH, PCBP1 and PCBP2, interact with HEV RNA promoter and RNA-dependent RNA polymerase to regulate HEV replication. We found that hnRNPK and hnRNPA2B1 are the virus-supportive factors interacting with HEV RNA at promoter regions along with HEV polymerase protein, which are essential for HEV replication in the cells. Contrarily, hnRNPH, PCBP1 and PCBP2 are the antiviral factors that interact exclusively with HEV genomic promoter and inhibit HEV replication in Huh7 S10-3 cells. In vitro RNA-binding assays revealed that the antiviral hnRNP proteins hamper the binding of virus-supportive hnRNP proteins at HEV genomic promoter. In the binding reaction, the binding of HEV polymerase protein to the genomic promoter is slightly affected by the presence of antiviral hnRNPH. In an effort of visualizing the subcellular localization of hnRNP proteins in the HEV replication scenario in the Huh7 cells, we showed that hnRNPK, hnRNPA2B1, hnRNPH, PCBP1 and PCBP2 redistribute from nucleus to cytoplasm. In conclusion, our study highlights the importance of hnRNP proteins in HEV replication regulation.

Hepatitis E virus polymerase binds to IFIT1 to protect the viral RNA from IFIT1-mediated translation inhibition.

Hepatitis E virus (HEV) induces interferons and regulates the induction of interferon-stimulated genes (ISGs) in the host cell. HEV infection has been shown to promote the expression of different ISGs, such as ISG15, IFIT1, MX1, RSAD2/Viperin and CxCL10, in cell culture and animal models. Interferon-induced protein with tetratricopeptide repeat 1 (IFIT1) is an ISG-encoded protein that inhibits the translation of viral RNA, having 5'-triphosphate or the mRNA lacking 2'-O-methylation on the 5'cap. In this study, we found that IFIT1 binds to HEV RNA to inhibit its translation. HEV replication is also restricted in hepatoma cells with overexpressed IFIT1. However, despite this binding of IFIT1 to HEV RNA, HEV successfully replicates in hepatoma cells in the infection scenario. In an effort to identify the underlying mechanism, we found that HEV RNA-dependent RNA polymerase (RdRp) binds to IFIT1, thereby protecting the viral RNA from IFIT1-mediated translation inhibition. RdRp sequesters IFIT1, resulting in the successful progression of viral replication in the infected cells. Thus, we discovered a distinct pro-viral role of HEV RdRp that is crucial for successful infection in the host, and propose a unique mechanism developed by HEV to overcome IFIT1-mediated host immune response.

Activities of Thrombin and Factor Xa Are Essential for Replication of Hepatitis E Virus and Are Possibly Implicated in ORF1 Polyprotein Processing.

Hepatitis E virus (HEV) is a clinically important positive-sense RNA virus. The ORF1 of HEV encodes a nonstructural polyprotein of 1,693 amino acids. It is not clear whether the ORF1 polyprotein (pORF1) is processed into distinct enzymatic domains. Many researchers have attempted to understand the mechanisms of pORF1 processing. However, these studies gave various results and could never convincingly establish the mechanism of pORF1 processing. In this study, we demonstrated the possible role of thrombin and factor Xa in pORF1 processing. We observed that the HEV pORF1 polyprotein bears conserved cleavage sites of thrombin and factor Xa. Using a reverse genetics approach, we demonstrated that an HEV replicon having mutations in the cleavage sites of either thrombin or factor Xa could not replicate efficiently in cell culture. Further, we demonstrated in vitro processing when we incubated recombinant pORF1 fragments with thrombin, and we observed the processing of pORF1 polyprotein. The treatment of a liver cell line with a serine protease inhibitor as well as small interfering RNA (siRNA) knockdown of thrombin and factor Xa resulted in significant reduction in the replication of HEV. Thrombin and factor Xa have been well studied for their roles in blood clotting. Both of these proteins are believed to be present in the active form in the blood plasma. Interestingly, in this report, we demonstrated the presence of biologically active thrombin and factor Xa in a liver cell line. The results suggest that factor Xa and thrombin are essential for the replication of HEV and may be involved in pORF1 polyprotein processing of HEV.IMPORTANCE Hepatitis E virus (HEV) causes a liver disorder called hepatitis in humans, which is mostly an acute and self-limiting infection in adults. A high mortality rate of about 30% is observed in HEV-infected pregnant women in developing countries. There is no convincing opinion about HEV ORF1 polyprotein processing owing to the variability of study results obtained so far. HEV pORF1 has cleavage sites for two host cellular serine proteases, thrombin and factor Xa, that are conserved among HEV genotypes. For the first time, this study demonstrated that thrombin and factor Xa cleavage sites on HEV pORF1 are obligatory for HEV replication. Intracellular biochemical activities of the said serine proteases are also essential for efficient HEV replication in cell culture and must be involved in pORF1 processing. This study sheds light on the presence and roles of clotting factors with respect to virus replication in the cells.