Udana Torian

A Bicistronic Subgenomic mRNA Encodes both the ORF2 and ORF3 Proteins of Hepatitis E Virus

ABSTRACT Hepatitis E virus replicons containing the neomycin resistance gene expressed from open reading frames (ORFs) 2 and 3 were transfected into Huh-7 cells, and stable cell lines containing functional replicons were selected by constant exposure to G418 sulfate. Northern blot analyses detected full-length replicon RNA and a single subgenomic RNA. This subgenomic RNA, which was capped, initiated at nucleotide 5122 downstream of the first two methionine codons in ORF3 and was bicistronic; two closely spaced methionine codons in different reading frames were used for the initiation of ORF3 and ORF2 translation.

Cross-species infections of cultured cells by hepatitis E virus and discovery of an infectious virus–host recombinant

The RNA virus, hepatitis E virus (HEV) is the most or second-most important cause of acute clinical hepatitis in adults throughout much of Asia, the Middle East, and Africa. In these regions it is an important cause of acute liver failure, especially in pregnant women who have a mortality rate of 20–30%. Until recently, hepatitis E was rarely identified in industrialized countries, but Hepatitis E now is reported increasingly throughout Western Europe, some Eastern European countries, and Japan. Most of these cases are caused by genotype 3, which is endemic in swine, and these cases are thought to be zoonotically acquired. However, transmission routes are not well understood. HEV that infect humans are divided into nonzoonotic (types 1, 2) and zoonotic (types 3, 4) genotypes. HEV cell culture is inefficient and limited, and thus far HEV has been cultured only in human cell lines. The HEV strain Kernow-C1 (genotype 3) isolated from a chronically infected patient was used to identify human, pig, and deer cell lines permissive for infection. Cross-species infections by genotypes 1 and 3 were studied with this set of cultures. Adaptation of the Kernow-C1 strain to growth in human hepatoma cells selected for a rare virus recombinant that contained an insertion of 174 ribonucleotides (58 amino acids) of a human ribosomal protein gene.

Adaptation of a Genotype 3 Hepatitis E Virus to Efficient Growth in Cell Culture Depends on an Inserted Human Gene Segment Acquired by Recombination

ABSTRACT An infectious cDNA clone of a genotype 3 strain of hepatitis E virus adapted to growth in HepG2/C3A human hepatoma cells was constructed. This virus was unusual in that the hypervariable region of the adapted virus contained a 171-nucleotide insertion that encoded 58 amino acids of human S17 ribosomal protein. Analyses of virus from six serial passages indicated that genomes with this insert, although initially rare, were selected during the first passage, suggesting it conferred a significant growth advantage. RNA transcripts from this cDNA and the viruses encoded by them were infectious for cells of both human and swine origin, the major host species for this zoonotic virus. Mutagenesis studies demonstrated that the S17 insert was a major factor in cell culture adaptation. Introduction of 54 synonymous mutations into the insert had no detectable effect, thus implicating protein, rather than RNA, as the important component. Truncation of the insert by 50% decreased the levels of successful transfection by ∼3-fold. Substitution of the S17 sequence by a different ribosomal protein sequence or by GTPase-activating protein sequence resulted in a partial enhancement of transfection levels, whereas substitution with 58 amino acids of green fluorescent protein had no effect. Therefore, both the sequence length and the amino acid composition of the insert were important. The S17 sequence did not affect transfection of human hepatoma cells when inserted into the hypervariable region of a genotype 1 strain, but this chimeric genome acquired a dramatic ability to replicate in hamster cells.

Release of Genotype 1 Hepatitis E Virus from Cultured Hepatoma and Polarized Intestinal Cells Depends on Open Reading Frame 3 Protein and Requires an Intact PXXP Motif

ABSTRACT Hepatitis E virus genotype 1 strain Sar55 replicated in subcloned Caco-2 intestinal cells and Huh7 hepatoma cells that had been transfected with in vitro transcribed viral genomes, and hepatitis E virions were released into the culture medium of both cell lines. Virus egress from cells depended on open reading frame 3 (ORF3) protein, and a proline-rich sequence in ORF3 was important for egress from cultured cells and for infection of macaques. Both intracellular ORF3 protein accumulation and virus release occurred at the apical membrane of polarized Caco-2 cells. ORF3 protein and lipids were intimately associated with virus particles produced in either cell line; ORF2 epitopes were masked in these particles and could not be immunoprecipitated with anti-ORF2.

ORF3 Protein of Hepatitis E Virus Is Not Required for Replication, Virion Assembly, or Infection of Hepatoma Cells In Vitro

ABSTRACT A subclone of Huh-7 cells that could be relatively efficiently transfected and infected with hepatitis E virus was identified. Following transfection, infectious virus was produced but remained predominantly cell associated. Intracellular virus, recovered by lysis of transfected cells, infected naïve cells. This in vitro-produced virus appeared to be antigenically identical to virus isolated from clinical samples. Lysates from cells transfected with mutant viral genomes unable to synthesize ORF3 protein contained infectious virions that were similar in number, thermostability, and sedimentation characteristics to those in lysates transfected with wild-type viral genomes. Therefore, in contrast to its requirement in vivo, ORF3 protein is not required for infection of Huh-7 cells or production of infectious virus in vitro.

Mutations within Potential Glycosylation Sites in the Capsid Protein of Hepatitis E Virus Prevent the Formation of Infectious Virus Particles

ABSTRACT Hepatitis E virus is a nonenveloped RNA virus. However, the single capsid protein resembles a typical glycoprotein in that it contains a signal sequence and potential glycosylation sites that are utilized when recombinant capsid protein is overexpressed in cell culture. In order to determine whether these unexpected observations were biologically relevant or were artifacts of overexpression, we analyzed capsid protein produced during a normal viral replication cycle. In vitro transcripts from an infectious cDNA clone mutated to eliminate potential glycosylation sites were transfected into cultured Huh-7 cells and into the livers of rhesus macaques. The mutations did not detectably affect genome replication or capsid protein synthesis in cell culture. However, none of the mutants infected rhesus macaques. Velocity sedimentation analyses of transfected cell lysates revealed that mutation of the first two glycosylation sites prevented virion assembly, whereas mutation of the third site permitted particle formation and RNA encapsidation, but the particles were not infectious. However, conservative mutations that did not destroy glycosylation motifs also prevented infection. Overall, the data suggested that the mutations were lethal because they perturbed protein structure rather than because they eliminated glycosylation.

A naturally occurring human/hepatitis E recombinant virus predominates in serum but not in faeces of a chronic hepatitis E patient and has a growth advantage in cell culture

Hepatitis E virus is the aetiological agent of acute hepatitis E, a self-limiting disease prevalent in developing countries. Molecular analysis of viral genomic RNA from a chronically infected patient confirmed the recent discovery that chronic infection correlated with extensive diversification of the virus quasispecies: the hypervariable region of some virus genomes in this USA patient contained large continuous deletions and a minor proportion of genomes in faeces and serum had acquired a mammalian sequence that encoded 39 aa of S19 ribosomal protein fused to the virus non-structural protein. Genomes with this insert were selected during virus passage in cultured cells to become the predominant species, suggesting that the inserted sequence promoted virus growth. The results demonstrated that hepatitis E virus can mutate dramatically during a prolonged infection and suggests it may be important to prevent or cure chronic infections before new variants with unpredictable properties arise.

Hepatitis E Virus Genotype 1 Infection of Swine Kidney Cells In Vitro Is Inhibited at Multiple Levels

ABSTRACT Genotype 1 hepatitis E viruses (HEVs) are restricted to primate hosts, whereas genotype 3 HEVs predominantly infect swine, in addition to primates. In order to identify possible determinants of the host range, infectious recombinant viruses and chimeras of a genotype 1 isolate and a genotype 3 isolate were compared for their ability to infect versus transfect cultured human HepG2/C3A cells and swine LLC-PK cells. The patterns of luciferase expression from virus replicons containing the Gaussia luciferase gene in place of the viral ORF2 or ORF3 genes demonstrated that translation of the ORF2 capsid gene of genotype 1 virus is severely inhibited in swine kidney cells compared to its translation in rhesus macaque kidney or human liver cells. Therefore, this virus may produce insufficient capsid protein for optimal assembly in swine cells. Infectivity assays with a virus containing a chimeric capsid protein confirmed that amino acids 456 to 605 of the virus capsid protein comprised the virus receptor-binding region and suggested that genotype 1 viruses may be prevented from infecting swine because genotype 1 viruses are unable to enter swine cells. Rhesus macaque cells appeared to be better than human cells for growing the genotype 1 virus. These cell and virus combinations may serve as a useful in vitro model with which to study determinants of the natural host range of this virus.

An essential RNA element resides in a central region of hepatitis E virus ORF2

Hepatitis E virus (genus Hepevirus, family Hepeviridae) is one of the most important causes of acute hepatitis in adults, particularly among pregnant women, throughout Asia and Africa where mortality rates can be 20–30 %. Hepatitis E virus has a single-stranded positive-sense RNA genome that contains three translated ORFs. The two 3′ ORFs are translated from a subgenomic RNA. Functional RNA elements have been identified in and adjacent to the genomic 5′ and 3′ UTRs and in and around the intergenic region. Here we describe an additional RNA element that is located in a central region of ORF2. The RNA element is predicted to fold into two highly conserved stem–loop structures, ISL1 and ISL2. Mutations that disrupt the predicted structures, without altering the encoded amino acid sequence, result in a drastic reduction in capsid protein synthesis. This indicates that the RNA element plays an important role in one of the early steps of virus replication. The structures were further investigated using a replicon that expresses Gaussia luciferase in place of the capsid protein. Single mutations in ISL2 severely reduced luciferase expression, but a pair of compensatory mutations that were predicted to restore the ISL2 structure, restored luciferase expression to near-WT levels, thus lending experimental support to the predicted structure. Nonetheless the precise role of the ISL1+ISL2 element remains unknown.

Structural studies of influenza virus RNPs by electron microscopy indicate molecular contortions within NP supra-structures

Ribonucleoprotein (RNP) complexes of influenza viruses are composed of multiple copies of the viral nucleoprotein (NP) that can form filamentous supra-structures. RNPs package distinct viral genomic RNA segments of different lengths into pleomorphic influenza virions. RNPs also function in viral RNA transcription and replication. Different RNP segments have varying lengths, but all must be incorporated into virions during assembly and then released during viral entry for productive infection cycles. RNP structures serve varied functions in the viral replication cycle, therefore understanding their molecular organization and flexibility is essential to understanding these functions. Here, we show using electron tomography and image analyses that isolated RNP filaments are not rigid helical structures, but instead display variations in lengths, curvatures, and even tolerated kinks and local unwinding. Additionally, we observed NP rings within RNP preparations, which were commonly composed of 5, 6, or 7 NP molecules and were of similar widths to filaments, suggesting plasticity in NP-NP interactions mediate RNP structural polymorphism. To demonstrate that NP alone could generate rings of variable oligomeric state, we performed 2D single particle image analysis on recombinant NP and found that rings of 4 and 5 protomers dominated, but rings of all compositions up to 7 were directly observed with variable frequency. This structural flexibility may be needed as RNPs carry out the interactions and conformational changes required for RNP assembly and genome packaging as well as virus uncoating.