- Volume 97, Issue 8, 2016

Capsids of numerous filamentous and rod-shaped plant viruses possess helical symmetry. In positive-stranded RNA viruses, helical capsids are typically composed of many identical subunits of the viral capsid protein (CP), encapsidating a molecule of viral genomic RNA. Current progress in structural studies of helical plant viruses has revealed differences between filamentous and rod-shaped viruses, both in structural folds of their CPs and in the interactions of CP molecules in their capsids. Many filamentous and rod-shaped viruses have functionally similar lateral inter-subunit contacts on the outer virion surface. Additionally, the extreme N-terminal CP region in filamentous viruses is intrinsically disordered. Taken together, the available data establish a link between the structural features of molecular interactions of CP molecules and the physical properties of helical virions ranging from rigidity to flexibility. Overall, the structure of helical plant viruses is significantly more labile than previously thought, often allowing structural transitions, remodelling and the existence of alternative structural forms of virions. These properties of virions are believed to be functionally significant at certain stages of the viral life cycle, such as during translational activation and cell-to-cell transport. In this review, we discuss structural and functional features of filamentous and rod-shaped virions, highlight their shared features and differences, and lay emphasis on the relationships between the molecular structure of viral capsids and their properties including virion shape, lability and capability of structural remodelling.

Clinical isolates of influenza virus produce pleomorphic virus particles, including extremely long filamentous virions. In contrast, strains of influenza that have adapted to laboratory growth typically produce only spherical virions. As a result, the filamentous phenotype has been overlooked in most influenza virus research. Recent advances in imaging and improved animal models have highlighted the distinct structure and functional relevance of filamentous virions. In this review we summarize what is currently known about these strikingly elongated virus particles and discuss their possible roles in clinical infections.

Bovine respiratory disease (BRD) is the most costly disease affecting the cattle industry. The pathogenesis of BRD is complex and includes contributions from microbial pathogens as well as host, environmental and animal management factors. In this study, we utilized viral metagenomic sequencing to explore the virome of nasal swab samples obtained from feedlot cattle with acute BRD and asymptomatic pen-mates at six and four feedlots in Mexico and the USA, respectively, in April–October 2015. Twenty-one viruses were detected, with bovine rhinitis A (52.7 %) and B (23.7 %) virus, and bovine coronavirus (24.7 %) being the most commonly identified. The emerging influenza D virus (IDV) tended to be significantly associated (P=0.134; odds ratio=2.94) with disease, whereas viruses commonly associated with BRD such as bovine viral diarrhea virus, bovine herpesvirus 1, bovine respiratory syncytial virus and bovine parainfluenza 3 virus were detected less frequently. The detection of IDV was further confirmed using a real-time PCR assay. Nasal swabs from symptomatic animals had significantly more IDV RNA than those collected from healthy animals (P=0.04). In addition to known viruses, new genotypes of bovine rhinitis B virus and enterovirus E were identified and a newly proposed species of bocaparvovirus, Ungulate bocaparvovirus 6, was characterized. Ungulate tetraparvovirus 1 was also detected for the first time in North America to our knowledge. These results illustrate the complexity of the virome associated with BRD and highlight the need for further research into the contribution of other viruses to BRD pathogenesis.

The cap binding domain of the polymerase basic 2 (PB2) subunit of influenza polymerases plays a critical role in mediating the ‘cap-snatching' mechanism by binding the 5′ cap of host pre-mRNAs during viral mRNA transcription. Monitoring variations in the PB2 protein is thus vital for evaluating the pathogenic potential of the virus. Based on selection pressure analysis of PB2 gene sequences of the pandemic H1N1 (pH1N1) viruses of the period 2009–2014, we identified a site, 344V/M, in the vicinity of the cap binding pocket showing evidence of adaptive evolution and another co-evolving residue, 354I/L, in close vicinity. Modelling of the three-dimensional structure of the pH1N1 PB2 cap binding domain, docking of the pre-mRNA cap analogue m7GTP and molecular dynamics simulation studies of the docked complexes performed for four PB2 variants observed showed that the complex possessing V344M with I354L possessed better ligand binding affinity due to additional hydrogen bond contacts between m7GTP and the key residues His432 and Arg355 that was attributed to a displacement of the 424 loop and a flip of the side chain of Arg355, respectively. The co-evolutionary mutations identified (V344M, I354L) were found to be established in the PB2 gene of the pH1N1 viral population over the period 2010–2014. The study demonstrates the molecular basis for the enhanced m7GTP ligand binding affinity with the 344M–354L synergistic combination in PB2. Furthermore, the insight gained into understanding the molecular mechanism of cap binding in pH1N1 viruses may be useful for designing novel drugs targeting the PB2 cap binding domain.

Influenza A viruses have the potential to cause pandemics due to the introduction of novel subtypes against which human hosts have little or no preexisting immunity. Such viruses may result from reassortment between human and animal influenza viruses. Recently, new influenza-like viruses were identified in bats, raising the concern for a new reservoir of potentially harmful influenza viruses that could form reassortants with categorized human influenza A viruses. However, until now, it has not been possible to generate a recombinant reassortant virus containing a single functional gene or domain from H17N10 that could propagate. Here, we demonstrate that a recombinant A/Puerto Rico/8/1934 (H1N1) virus with NS1 gene from H17N10 influenza-like virus can be successfully rescued. We used luciferase reporter assays and quantitative reverse transcriptase PCR to show that the NS1 protein from H17N10 inhibited Sendai-virus (SeV)-induced activation of IFN-β expression with an efficiency similar to NS1 from an H5N1 strain. Moreover, the crystal structure of the NS1 (H17N10) RNA-binding domain is also similar to that of other NS1s. These results demonstrate that H17N10 influenza-like virus indeed contains functional genes that are compatible with categorized influenza A viruses. Although the chance of this particular event occurring in nature seems negligible, further research is needed to address the possibility of the natural formation of reassortants.

Immunomodulators have been shown to improve the outcome of severe pneumonia. We have previously shown that mycophenolic acid (MPA), an immunomodulator, has antiviral activity against influenza A/WSN/1933(H1N1) using a high-throughput chemical screening assay. This study further investigated the antiviral activity and mechanism of action of MPA against contemporary clinical isolates of influenza A and B viruses. The 50 % cellular cytotoxicity (CC50) of MPA in Madin Darby canine kidney cell line was over 50 µM. MPA prevented influenza virus-induced cell death in the cell-protection assay, with significantly lower IC50 for influenza B virus B/411 than that of influenza A(H1N1)pdm09 virus H1/415 (0.208 vs 1.510 µM, P=0.0001). For H1/415, MPA interfered with the early stage of viral replication before protein synthesis. For B/411, MPA may also act at a later stage since MPA was active against B/411 even when added 12 h post-infection. Virus-yield reduction assay showed that the replication of B/411 was completely inhibited by MPA at concentrations ≥0.78 µM, while there was a dose-dependent reduction of viral titer for H1/415. The antiviral effect of MPA was completely reverted by guanosine supplementation. Plaque reduction assay showed that MPA had antiviral activity against eight different clinical isolates of A(H1N1), A(H3N2), A(H7N9) and influenza B viruses (IC50 <1 µM). In summary, MPA has broad-spectrum antiviral activity against human and avian-origin influenza viruses, in addition to its immunomodulatory activity. Together with a high chemotherapeutic index, the use of MPA as an antiviral agent should be further investigated in vivo.

Ljungan virus (LV) has been isolated/detected from rodents in a limited area including European countries and the USA. In this study, we isolated an LV strain from faecal samples of wild birds that had been collected in Japan, and determined the nearly complete sequence of the genome. Sequence analyses showed that the isolate possesses an LV-like genomic organization: 5UTR-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3UTR. Phylogenetic and similarity analyses based on the VP1 region indicated that the strain constitutes a novel genotype within LV. In addition, we identified species origin of the faeces as gull species by using the DNA barcoding technique. These data suggested that the novel LV strain infected a gull species, in which the virus had not been identified. Taken together, this study has provided the first evidence of the presence of a novel LV in Japan, highlighting the possibility of LV infection in birds.

We previously identified a third porcine epidemic diarrhoea virus (PEDV) S variant with a large deletion of 582 nucleotides in the 5′ terminal region of the S gene, in addition to the North American type and the S INDELs type. To investigate the pathogenicity of this variant, TTR-2/JPN/2014, we performed experimental infection using colostrum-deprived piglets and compared the results with those from the North American type PEDV, OKN-1/JPN/2013. Fifteen newborn piglets were divided into two groups of 7–8 piglets each and inoculated orally with one of PEDV isolates maintained at the eighth passage in Vero cell culture. Although all PEDV-inoculated piglets showed acute watery diarrhoea, lethality clearly differed between both PEDV-inoculated groups. Moreover, there were differences in virus distribution and lesions on the intestines between the two PEDV-inoculated groups. Therefore, our data suggest that the OKN-1/JPN/2013 PEDV isolate is virulent, whereas the TTR-2/JPN/2014 PEDV isolate is avirulent.

Hepatitis E virus (HEV) is a causative agent of acute hepatitis and a major public health problem in India. There are four mammalian HEV genotypes worldwide. In India, genotype 1 (HEV-1) is restricted to humans whereas genotype 4 (HEV-4) circulates in pigs. Studies from our laboratory have shown that HEV-4 (swine) virus can establish experimental infection in rhesus monkeys; however, HEV-1 (human) virus cannot infect pigs. Viral and/or cellular factors responsible for this host specificity are not yet known. We developed 12 different genotype 1–4 chimeric full genome clones with pSK-HEV2 as the backbone and by replacing structural (ORF2 and ORF3), non-structural (ORF1) and non-coding regions (NCR) with corresponding segments from the HEV-4 clone. S10-3 (human hepatoma) and PK-15 (pig kidney) cells were transfected with transcripts generated from the above clones to test their replication competence. Transfected cells were monitored for successful virus replication by detecting replicative intermediate RNA and capsid protein (immunofluorescence assay). All the chimeric constructs were able to replicate in S10-3 cells. However, only two chimeric clones, HEV-1 (HEV-4 5′NCR-ORF1) and HEV-1 (HEV-4 ORF1), containing 5′NCR-ORF1 and ORF1 regions from the HEV-4 clone, respectively, were able to replicate in PK-15 cells. We demonstrate for the first time the crucial role of ORF1 polyprotein in crossing the species barrier at the cellular level. These results indicate the importance of interactions between ORF1 protein domains and host cell specific factors during HEV replication and the critical role of cellular factors as post-entry barrier/s in virus establishment.

Picornaviruses form replication complexes in association with membranes in structures called replication organelles. Common themes to emerge from studies of picornavirus replication are the need for cholesterol and phosphatidylinositol 4-phosphate (PI4P). In infected cells, type III phosphatidylinositol 4-kinases (PI4KIIIs) generate elevated levels of PI4P, which is then exchanged for cholesterol at replication organelles. For the enteroviruses, replication organelles form at Golgi membranes in a process that utilizes PI4KIIIβ. Other picornaviruses, for example the cardioviruses, are believed to initiate replication at the endoplasmic reticulum and subvert PI4KIIIα to generate PI4P. Here we investigated the role of PI4KIII in foot-and-mouth disease virus (FMDV) replication. Our results showed that, in contrast to the enteroviruses and the cardioviruses, FMDV replication does not require PI4KIII (PI4KIIIα and PI4KIIIβ), and PI4P levels do not increase in FMDV-infected cells and PI4P is not seen at replication organelles. These results point to a unique requirement towards lipids at the FMDV replication membranes.

The cytoplasmic tails of some coronavirus (CoV) spike (S) proteins contain an endoplasmic reticulum retrieval signal (ERRS) that can retrieve S proteins from the Golgi to the endoplasmic reticulum (ER); this process is thought to accumulate S proteins at the CoV budding site, the ER-Golgi intermediate compartment (ERGIC), and to facilitate S protein incorporation into virions. However, we showed previously that porcine epidemic diarrhoea CoV S proteins lacking the ERRS were efficiently incorporated into virions, similar to the original virus. Thus, the precise role of the ERRS in virus assembly remains unclear. Here, the roles of the S protein ERRS in severe acute respiratory syndrome CoV (SARS-CoV) intracellular trafficking and S incorporation into virus-like particles (VLPs) are described. Intracellular trafficking and indirect immunofluorescence analysis suggested that when M protein was present, wild-type S protein (wtS) could be retained in the pre- and post-medial Golgi compartments intracellularly and co-localized with M protein in the Golgi. In contrast, mutant S protein lacking the ERRS was distributed throughout the ER and only partially co-localized with M protein. Moreover, the intracellular accumulation of mutant S protein, particularly at the post-medial Golgi compartment, was significantly reduced compared with wtS. A VLP assay suggested that wtS that reached the post-medial compartment could be returned to the ERGIC for subsequent incorporation into VLPs, while mutant S protein could not. These results suggest that the ERRS of SARS-CoV contributes to intracellular S protein accumulation specifically in the post-medial Golgi compartment and to S protein incorporation into VLPs.

An effective immune response against hepatitis C virus (HCV) requires the early development of multi-specific class 1 CD8+ and class II CD4+ T-cells together with broad neutralizing antibody responses. We have produced mammalian-cell-derived HCV virus-like particles (VLPs) incorporating core, E1 and E2 of HCV genotype 1a to produce such immune responses. Here we describe the biochemical and morphological characterization of the HCV VLPs and study HCV core-specific T-cell responses to the particles. The E1 and E2 glycoproteins in HCV VLPs formed non-covalent heterodimers and together with core protein assembled into VLPs with a buoyant density of 1.22 to 1.28 g cm−3. The HCV VLPs could be immunoprecipited with anti-ApoE and anti-ApoC. On electron microscopy, the VLPs had a heterogeneous morphology and ranged in size from 40 to 80 nm. The HCV VLPs demonstrated dose-dependent binding to murine-derived dendritic cells and the entry of HCV VLPs into Huh7 cells was blocked by anti-CD81 antibody. Vaccination of BALB/c mice with HCV VLPs purified from iodixanol gradients resulted in the production of neutralizing antibody responses while vaccination of humanized MHC class I transgenic mice resulted in the prodution of HCV core-specific CD8+ T-cell responses. Furthermore, IgG purified from the sera of patients chronically infected with HCV genotypes 1a and 3a blocked the binding and entry of the HCV VLPs into Huh7 cells. These results show that our mammalian-cell-derived HCV VLPs induce humoral and HCV-specific CD8+ T-cell responses and will have important implications for the development of a preventative vaccine for HCV.

The hepatitis C virus (HCV) RNA genome of 9.6 kb encodes only 10 proteins, and so is highly dependent on host hepatocyte factors to facilitate replication. We aimed to identify host factors involved in the egress of viral particles. By screening the supernatant of HCV-infected Huh7 cells using SILAC-based proteomics, we identified the transmembrane protein calsyntenin-1 as a factor specifically secreted by infected cells. Calsyntenin-1 has previously been shown to mediate transport of endosomes along microtubules in neurons, through interactions with kinesin light chain-1. Here we demonstrate for the first time, we believe, a similar role for calsyntenin-1 in Huh7 cells, mediating intracellular transport of endosomes. In HCV-infected cells we show that calsyntenin-1 contributes to the early stages of the viral replication cycle and the formation of the replication complex. Importantly, we demonstrate in our model that silencing calsyntenin-1 disrupts the viral replication cycle, confirming the reliance of HCV on this protein as a host factor. Characterizing the function of calsyntenin-1 will increase our understanding of the HCV replication cycle and pathogenesis, with potential application to other viruses sharing common pathways.

Group C rotaviruses (RVC) are enteric pathogens of humans and animals. Whole-genome sequences are available only for few RVCs, leaving gaps in our knowledge about their genetic diversity. We determined the full-length genome sequence of two human RVCs (PR2593/2004 and PR713/2012), detected in Italy from hospital-based surveillance for rotavirus infection in 2004 and 2012. In the 11 RNA genomic segments, the two Italian RVCs segregated within separate intra-genotypic lineages showed variation ranging from 1.9 % (VP6) to 15.9 % (VP3) at the nucleotide level. Comprehensive analysis of human RVC sequences available in the databases allowed us to reveal the existence of at least two major genome configurations, defined as type I and type II. Human RVCs of type I were all associated with the M3 VP3 genotype, including the Italian strain PR2593/2004. Conversely, human RVCs of type II were all associated with the M2 VP3 genotype, including the Italian strain PR713/2012. Reassortant RVC strains between these major genome configurations were identified. Although only a few full-genome sequences of human RVCs, mostly of Asian origin, are available, the analysis of human RVC sequences retrieved from the databases indicates that at least two intra-genotypic RVC lineages circulate in European countries. Gathering more sequence data is necessary to develop a standardized genotype and intra-genotypic lineage classification system useful for epidemiological investigations and avoiding confusion in the literature.

Human herpesvirus 6 (HHV-6) can integrate its genome into the telomeres of host chromosomes and is present in the germline of about 1 % of the human population. HHV-6 encodes a putative integrase U94 that possesses all molecular functions required for recombination including DNA-binding, ATPase, helicase and nuclease activity, and was hypothesized by many researchers to facilitate integration ever since the discovery of HHV-6 integration. However, analysis of U94 in the virus context has been hampered by the lack of reverse-genetic systems and efficient integration assays. Here, we addressed the role of U94 and the cellular recombinase Rad51 in HHV-6 integration. Surprisingly, we could demonstrate that HHV-6 efficiently integrated in the absence of U94 using a new quantitative integration assay. Additional inhibition of the cellular recombinase Rad51 had only a minor impact on virus integration. Our results shed light on this complex integration mechanism that includes factors beyond U94 and Rad51.

The coupling of mRNA processing steps is essential for precise and efficient gene expression. The human transcription/export (hTREX) complex is a highly conserved multi-protein complex responsible for eukaryotic mRNA stability and nuclear export. We have previously shown that the Kaposi’s sarcoma-associated open reading frame 57 (ORF57) protein orchestrates the recruitment of the hTREX complex onto viral intronless mRNA, forming a stable and export-competent viral ribonucleoprotein particle (vRNP). Recently, additional cellular proteins, namely CHTOP, CIP29 and POLDIP3 have been proposed as novel hTREX components. Herein, we extend our previous research and provide evidence that ORF57 interacts with CHTOP and CIP29, in contrast to POLDIP3. Moreover, depletion studies show both CHTOP and CIP29 effect ORF57-mediated viral mRNA processing. As such, these results suggest both CHTOP and CIP29 are hTREX components and are recruited to an ORF57-mediated vRNP.

Recent studies have generated interest in the function of human adenovirus serotype 5 (HAdV-5) hexon:  factor X (FX) binding and subsequent hepatocyte transduction and interaction with the immune system. Here, we retargeted adenovirus serotype 5 vectors, ablated for FX interaction, by replacing amino acids in hexon HVR7 with RGD-4C or inserting the peptide into the fibre HI loop. These genetic modifications in the capsid were compatible with virus assembly, and could efficiently retarget transduction of the vector via the αvβ3/5 integrin-mediated pathway, but did not alter immune recognition by pre-existing human neutralizing anti-HAdV-5 antibodies or by natural antibodies in mouse serum. Thus, FX-binding-ablated HAdV-5 can be retargeted but remain sensitive to immune-mediated attack. These findings further refine HAdV-5-based vectors for human gene therapy and inform future vector development.

The glycoproteins gH and gL of human cytomegalovirus (HCMV) form a complex either with pUL74 (trimeric complex) or with proteins of the UL128 locus (pentameric complex). While the pentameric complex is dispensable for viral growth in fibroblasts, deletion of pUL74 causes a small plaque phenotype in HCMV lab strains, accompanied by greatly reduced cell-free infectivity. As HCMV isolates, shortly after cultivation from clinical specimens, do not release cell-free infectious viruses, we wondered whether deletion of pUL74 would also affect virus growth in this background. To address this question, we took advantage of the bacterial artificial chromosome (BAC)-cloned virus Merlin-RL13tetO, which grows cell associated due to the inducible expression of the viral RL13 gene, thereby resembling clinical isolates. Stop codons were introduced by seamless mutagenesis into UL74 and/or the UL128 locus to prevent expression of the trimeric or pentameric complex, respectively. Virus mutants were reconstituted by transfection of the respective genomes into cultured cells and analysed with respect to focal growth. When the UL128 locus was intact, deletion of pUL74 did not notably affect focal growth of Merlin, irrespective of RL13 expression. In the absence of UL128 expression, foci were increased compared with wild-type, and infectious cell-free virus was produced. Under these conditions, disruption of UL74 completely prevented virus spread from initially transfected cells to surrounding cells. In conclusion the contribution of pUL74 is masked when the UL128 locus is expressed at high levels, and its role in cell-free virus spread is only revealed when expression of the pentameric complex is inhibited.