The Lysine Residues within the Human Ribosomal Protein S17 Sequence Naturally Inserted into the Viral Nonstructural Protein of a Unique Strain of Hepatitis E Virus Are Important for Enhanced Virus Replication

Abstract

Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Due largely to the lack of an efficient cell culture system for HEV, the molecular mechanisms of HEV replication and pathogenesis are poorly understood. Recently, a unique genotype 3 strain of HEV recovered from a chronically infected patient was adapted for growth in HepG2C3A human hepatoma cells. The adaptation of the Kernow C-1 P6 HEV to propagate in HepG2C3A cells selected for a rare virus recombinant that contains an insertion of a 171-nucleotide sequence encoding amino acids 21 to 76 of the human ribosomal protein S17 (RPS17) within the hypervariable region (HVR) of the HEV ORF1 protein. When the RPS17 insertion was placed into a strain of genotype 1 HEV which infects only humans, it expanded the host range of the virus, allowing it to infect cell lines from multiple animal species, including cow, dog, cat, chicken, and hamster. In this study, we utilized forward and reverse genetics to attempt to define which aspects of the RPS17 insertion allow for the ability of the Kernow C-1 P6 HEV to adapt in cell culture and allow for expanded host tropism. We demonstrate that the RPS17 sequence insertion in HEV bestows novel nuclear/nucleolar trafficking capabilities to the ORF1 protein of Kernow P6 HEV and that lysine residues within the RPS17 insertion, but not nuclear localization of the ORF1 protein, correlate with the enhanced replication of the HEV Kernow C-1 P6 strain. The results from this study have important implications for understanding the mechanism of cross-species infection and replication of HEV. IMPORTANCEHEV is an important pathogen worldwide. The virus causes high mortality (up to 30%) in pregnant women and has been recognized to cause chronic hepatitis in immunocompromised populations. The life cycle of HEV has been understudied due to a lack of sufficient cell culture systems in which to propagate the virus. Recently, insertions and rearrangements of the hypervariable region (HVR) within the HEV genome, allowing for cell culture adaptation and expansion of the host range, have been reported. We utilized these cell culture-adapted HEV strains to assess how the HVR may be involved in virus replication and host range. We provide evidence that insertion of the RPS17 sequence in HEV likely confers nuclear trafficking capabilities to the nonstructural protein of the virus and that lysine residues within the RPS17 insertion are important for enhanced replication of the virus. These data will help to elucidate the mechanism of cross-species infection of HEV in the future. Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis in many parts of the world (1, 2). Major outbreaks and epidemics are most common in developing countries, where a lack of proper sanitation conditions leads to waterborne spread of the disease. In industrialized countries, HEV infection is typically sporadic and endemic. Recently, chronic HEV infection has become an important clinical problem in immunosuppressed individual...

Evidence for an unknown agent antigenically related to the hepatitis E virus in dairy cows in the United States

“…Afterward, the samples were removed, the cells were washed with PBS, and fresh DMEM medium was added and incubated for 5 to 6 days. The cells were stained at 5 to 6 days after infection by the immunofluorescence assay with a rabbit antisera against a bacterially derived 6x His capsid protein containing a 111 N‐terminal amino acid truncation from the HEV Kernow‐C1‐P6 strain as the primary antibody and Alexa Fluor 488 goat anti‐rabbit IgG (H&L) (Molecular Probes, Life Technologies, Carlsbad, CA) as the secondary antibody. The number of positive cells was counted and recorded as the number of fluorescein focus units.…”

Regulation of Ribosomal Proteins on Viral Infection

“…RPL40 is required for cap-dependent translation initiation of Vesicular stomatitis virus, Measles virus and Rabies virus by acting in the 80S formation step [8]. A Hepatitis E virus recombinant strain with RPS17 insertion in the hypervariable region of viral ORF1 endows enhanced virus replication activity, indicating the importance of RPS17 for virus infection [101]. The interaction of eIF-5A with HIV-1 Rev trans-activator mediates the nuclear export of viral unspliced mRNA [102].…”

The non-structural protein Nsp2TF of porcine reproductive and respiratory syndrome virus down-regulates the expression of Swine Leukocyte Antigen class I

“…The expression of Nsp2 and Nsp2TF in transfected cells was determined by an IFA as described previously (Kenney and Meng, 2015). Briefly, cells transfected with Nsp2 or Nsp2TF constructs were fixed at 24 h post-transfection with a 4% paraformaldehyde Then the fixed cells were incubated with a polyclonal antibody against Nsp2TF or Nsp2 (1:1000) previously mentioned at 37°C for 1 h. After extensive washing with PBS, cells were incubated with Alexa fluor594-conjugated goat anti-rabbit IgG (1:1000) for 1 h at 37°C.…”