Early Interaction of Rhinoviruses with Host Cells

The rate of attachment of type 2 virions to suspensions of HeLa cells is much greater than that of type 14, but the number of receptor sites per cell is similar for each type. The receptor sites may be partly saturated with excess virions; attachment is greatly reduced after about 10(4) particles have been taken up per cell. A lack of saturation of type 14 receptors by excess type 2 indicates that their receptor sites are separate on the cell surface. Excess of type 2 blocks attachment of type 1A, however, and excess of type 14 blocks type 51. Attachment of the human rhinoviruses is temperature-dependent with a Q(10) of 2.7. The eclipse reaction is also temperature-dependent. At 34.5 C, the irreversible eclipse of cell-associated rhinovirus type 2 requires only a few minutes, whereas the rate of eclipse of cell-associated type 14 is considerably slower. The eclipse product of type 2 rhinovirus has been recovered from infected cells. It sediments at about 90% of the rate of the infective virions and is missing virus polypeptide 4 (the smallest of the capsid polypeptides). Upon being subjected to CsCl gradient centrifugation, virus polypeptide 2 is also lost but the product still contains ribonucleic acid and bands at about 1.45 g/cc.     

In Vitro Survey of Autographa californica Nuclear Polyhedrosis Virus Interaction with Nontarget Vertebrate Host Cells

Thirty-five nontarget host cell lines, 23 of human and 12 of nonhuman vertebrate origin, were exposed to Autographa californica nuclear polyhedrosis virus preparations derived from four different sources: polyhedra, hemolymph, cell culture medium, and cultured cells. The virus and cells were incubated together at two different temperatures, 28 or 37°C, for four different lengths of time, 16, 40, 64, or 168 h, and the cells were assayed for the presence of virus by a peroxidase-antiperoxidase detection method. The estimated sensitivity of the assay as routinely conducted was 0.98 ng of alkali-liberated viral protein and 1.95 ng of budded viral protein per mm². No evidence of frank replication was obtained in any of the 35 cell lines tested, although virus uptake appeared to be quite common. Virus uptake was confirmed in some cases by electron microscopy. The degree of virus uptake appeared to be dependent on cell type, time and temperature of incubation, and viral phenotype. Virus purified from polyhedra was generally taken up more readily than were the other forms tested.     

Heterokaryon Formation of Simian Virus 40-transformed Cells in the Presence of Ultraviolet-irradiated Sendai Virus

Most simian virus 40-transformed mouse kidney lines form heterokaryons with CV-1 cells in the presence of ultraviolet-irradiated Sendai. However, two nonyielder lines, mKS-U2 and mKS-U20, fuse poorly.     

Porcine Encephalomyocarditis Virus Persists in Pig Myocardium and Infects Human Myocardial Cells

Recent advances toward using pig tissues in human transplantation have made it necessary to determine the risk of transmitting zoonotic viruses from pigs to humans or vice versa. We investigated the suitability of the porcine encephalomyocarditis virus (EMCV) model for such studies by determining its ability to persist in pigs, escape detection by routine serological methods, and infect human cells. Intraperitoneal inoculation of 5-week-old pigs with EMCV-30, a strain isolated from commercial pigs, resulted in acute cellular degeneration, infiltration of lymphocytes, and apoptosis in myocardium in 13 of 15 (86.7%) pigs during the acute phase of disease (3 to 21 days postinfection), followed by less-severe lymphocytic infiltration and apoptosis in 5 of 10 (50%) pigs during the chronic phase of the disease (day 45 to 90 postinfection). In the brain, lymphocytic infiltration, neuronal degeneration, and gliosis were observed in 26 to 33% of pigs in the acute phase of disease whereas perivascular cuffing was the predominant feature during chronic disease. EMCV antigens and RNA were demonstrated in the myocardium and brain during the chronic phase of disease. Analysis of 100 commercial pigs that were negative for EMCV antibodies identified two pig hearts positive for EMCV RNA. Porcine EMCV productively infected primary human cardiomyocytes as demonstrated by immunostaining using a monoclonal antibody specific for EMCV RNA polymerase, which is expressed only in productively infected cells, and by a one-step growth curve that showed production of 100 to 1,000 PFU of virus per cell within 6 h. The findings that porcine EMCV can persist in pig myocardium and can infect human myocardial cells make it an important infectious agent to screen for in pig-to-human cardiac transplants and a good model for xenozoonosis.     

Encephalomyocarditis Virus Infection of Mouse Plasmacytoma Cells I. Inhibition of Cellular Protein Synthesis

Mouse plasmacytoma ascites tumor cells (MOPC 460) were efficiently infected with encephalomyocarditis virus. Inhibition of host protein synthesis was evident after 2 h and complete by 4 h postinfection. The mechanism by which virus infection results in inhibition of host cell protein synthesis was studied in vitro. Cell-free protein-synthesizing systems, prepared from uninfected and infected cells, were found to be equally active with respect to their abilities to translate cellular and viral mRNAs. The plasmacytoma cell-free system was also shown to be insensitive to the addition of double-stranded viral RNA. Host cellular mRNA was isolated from uninfected and infected cells. No difference in the amount or size distribution of the mRNA was detected. However, the mRNA from infected cells was translated only 46 to 49% as actively as that from uninfected cells. mRNA isolated from cells in which initiation of protein synthesis was inhibited with pactamycin was similarly inactivated. Simultaneous addition of viral RNA and cellular mRNA to the plasmacytoma cell-free system resulted in a complete suppression of the translation of the cellular message, whereas viral RNA was translated normally.     

Variation in RNA Virus Mutation Rates across Host Cells

It is well established that RNA viruses exhibit higher rates of spontaneous mutation than DNA viruses and microorganisms. However, their mutation rates vary amply, from 10⁻⁶ to 10⁻⁴ substitutions per nucleotide per round of copying (s/n/r) and the causes of this variability remain poorly understood. In addition to differences in intrinsic fidelity or error correction capability, viral mutation rates may be dependent on host factors. Here, we assessed the effect of the cellular environment on the rate of spontaneous mutation of the vesicular stomatitis virus (VSV), which has a broad host range and cell tropism. Luria-Delbrück fluctuation tests and sequencing showed that VSV mutated similarly in baby hamster kidney, murine embryonic fibroblasts, colon cancer, and neuroblastoma cells (approx. 10⁻⁵ s/n/r). Cell immortalization through p53 inactivation and oxygen levels (1–21%) did not have a significant impact on viral replication fidelity. This shows that previously published mutation rates can be considered reliable despite being based on a narrow and artificial set of laboratory conditions. Interestingly, we also found that VSV mutated approximately four times more slowly in various insect cells compared with mammalian cells. This may contribute to explaining the relatively slow evolution of VSV and other arthropod-borne viruses in nature.     

Modulation of the Host Lipid Landscape to Promote RNA Virus Replication: The Picornavirus Encephalomyocarditis Virus Converges on the Pathway Used by Hepatitis C Virus

Cardioviruses, including encephalomyocarditis virus (EMCV) and the human Saffold virus, are small non-enveloped viruses belonging to the Picornaviridae, a large family of positive-sense RNA [(+)RNA] viruses. All (+)RNA viruses remodel intracellular membranes into unique structures for viral genome replication. Accumulating evidence suggests that picornaviruses from different genera use different strategies to generate viral replication organelles (ROs). For instance, enteroviruses (e.g. poliovirus, coxsackievirus, rhinovirus) rely on the Golgi-localized phosphatidylinositol 4-kinase III beta (PI4KB), while cardioviruses replicate independently of the kinase. By which mechanisms cardioviruses develop their ROs is currently unknown. Here we show that cardioviruses manipulate another PI4K, namely the ER-localized phosphatidylinositol 4-kinase III alpha (PI4KA), to generate PI4P-enriched ROs. By siRNA-mediated knockdown and pharmacological inhibition, we demonstrate that PI4KA is an essential host factor for EMCV genome replication. We reveal that the EMCV nonstructural protein 3A interacts with and is responsible for PI4KA recruitment to viral ROs. The ensuing phosphatidylinositol 4-phosphate (PI4P) proved important for the recruitment of oxysterol-binding protein (OSBP), which delivers cholesterol to EMCV ROs in a PI4P-dependent manner. PI4P lipids and cholesterol are shown to be required for the global organization of the ROs and for viral genome replication. Consistently, inhibition of OSBP expression or function efficiently blocked EMCV RNA replication. In conclusion, we describe for the first time a cellular pathway involved in the biogenesis of cardiovirus ROs. Remarkably, the same pathway was reported to promote formation of the replication sites of hepatitis C virus, a member of the Flaviviridae family, but not other picornaviruses or flaviviruses. Thus, our results highlight the convergent recruitment by distantly related (+)RNA viruses of a host lipid-modifying pathway underlying formation of viral replication sites.     

Differential Proteome Analysis of Chikungunya Virus Infection on Host Cells

Background

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus that has caused multiple unprecedented and re-emerging outbreaks in both tropical and temperate countries. Despite ongoing research efforts, the underlying factors involved in facilitating CHIKV replication during early infection remains ill-characterized. The present study serves to identify host proteins modulated in response to early CHIKV infection using a proteomics approach.

Methodology and Principal Findings

The whole cell proteome profiles of CHIKV-infected and mock control WRL-68 cells were compared and analyzed using two-dimensional gel electrophoresis (2-DGE). Fifty-three spots were found to be differentially modulated and 50 were successfully identified by MALDI-TOF/TOF. Eight were significantly up-regulated and 42 were down-regulated. The mRNA expressions of 15 genes were also found to correlate with the corresponding protein expression. STRING network analysis identified several biological processes to be affected, including mRNA processing, translation, energy production and cellular metabolism, ubiquitin-proteasome pathway (UPP) and cell cycle regulation.

Conclusion/Significance

This study constitutes a first attempt to investigate alteration of the host cellular proteome during early CHIKV infection. Our proteomics data showed that during early infection, CHIKV affected the expression of proteins that are involved in mRNA processing, host metabolic machinery, UPP, and cyclin-dependent kinase 1 (CDK1) regulation (in favour of virus survival, replication and transmission). While results from this study complement the proteomics results obtained from previous late host response studies, functional characterization of these proteins is warranted to reinforce our understanding of their roles during early CHIKV infection in humans.     

Gene Order of Encephalomyocarditis Virus as Determined by Studies with Pactamycin

Previous work has shown that translation of the encephalomyocarditis (EMC) viral ribonucleic acid (RNA) generates at least three primary products, polypeptides A, F, and C. The A and C polypeptides then undergo post-translational cleavages to complete the production of the stable viral polypeptides (delta, beta, gamma, alpha, G, I, F, H, and E). In this communication we show that A, F, and C are produced in equimolar amounts giving further support to the theory that the RNA of picornaviruses has only a single site for the initiation of protein synthesis. The biosynthesis of viral proteins in EMC virus-infected HeLa cells was studied in the presence of pactamycin at concentrations which preferentially inhibit the initiation of protein synthesis. The amount of each polypeptide formed during the residual period of protein synthesis observed after the addition of pactamycin was used as a criterion for ordering the genes on the viral RNA. The results obtained indicate that the primary gene products are ordered on the EMC viral RNA 5' --> 3' A-F-C and that the stable products are ordered delta-beta-gamma-alpha-G-I-F-H-E. Moreover, the intermediate chains B and epsilon map in the capsid region, whereas the intermediate chain D maps in the E region. This order is largely consistent with previously established relationships of the viral polypeptides and thus indicates that pactamycin is a valid tool for "genetic" mapping of polycistronic RNA molecules with single initiation sites.     

Fusion of Tumour Cells with Host Cells

THE A9 cell is an 8-azaguanine-resistant derivative of the L cell line¹. It lacks the enzyme inosinic acid pyrophosphorylase and is thus unable to grow in media such as HAT² in which endogenous synthesis of nucleic acid is blocked by aminopterin. The A9 line has little ability to grow progressively in vivo. Inocula of 5 × 10⁴ to 2 × 10⁶ cells produced progressive tumours in only 12% of X-irradiated newborn syngeneic C3H mice³. One of these tumours was explanted as a cell suspension into Eagle's minimal essential medium containing 15% foetal calf serum and then subcultivated in this medium with 5% foetal calf serum. At each passage, cells were inoculated into X-irradiated newborn syngeneic C3H or semi-allogeneic C3H×X F₁ mice (X designates a number of different allogeneic parents). Between 80 and 90% of the inoculated animals developed progressive tumours. The cell line was therefore designated A9HT (high take incidence). The karyotype of the A9HT line was found to be similar to that of the A9 line, but with a slightly reduced total chromosome number. The modal chromosome number of A9HT was about 53, compared with about 57 for A9 (see ref. 4). A9 and A9HT both had between 20 and 30 bi-armed chromosomes and a number of marker chromosomes in common. A detailed comparison of the karyotypes of the two lines examined by the quinacrine fluorescence technique has been made⁵. The A9HT line, like its A9 parent, lacks inosinic acid pyrophos-phorylase and is unable to grow in HAT medium.     

African Swine Fever Virus Uses Macropinocytosis to Enter Host Cells

African swine fever (ASF) is caused by a large and highly pathogenic DNA virus, African swine fever virus (ASFV), which provokes severe economic losses and expansion threats. Presently, no specific protection or vaccine against ASF is available, despite the high hazard that the continued occurrence of the disease in sub-Saharan Africa, the recent outbreak in the Caucasus in 2007, and the potential dissemination to neighboring countries, represents. Although virus entry is a remarkable target for the development of protection tools, knowledge of the ASFV entry mechanism is still very limited. Whereas early studies have proposed that the virus enters cells through receptor-mediated endocytosis, the specific mechanism used by ASFV remains uncertain. Here we used the ASFV virulent isolate Ba71, adapted to grow in Vero cells (Ba71V), and the virulent strain E70 to demonstrate that entry and internalization of ASFV includes most of the features of macropinocytosis. By a combination of optical and electron microscopy, we show that the virus causes cytoplasm membrane perturbation, blebbing and ruffles. We have also found that internalization of the virions depends on actin reorganization, activity of Na⁺/H⁺ exchangers, and signaling events typical of the macropinocytic mechanism of endocytosis. The entry of virus into cells appears to directly stimulate dextran uptake, actin polarization and EGFR, PI3K-Akt, Pak1 and Rac1 activation. Inhibition of these key regulators of macropinocytosis, as well as treatment with the drug EIPA, results in a considerable decrease in ASFV entry and infection. In conclusion, this study identifies for the first time the whole pathway for ASFV entry, including the key cellular factors required for the uptake of the virus and the cell signaling involved.     

Low-Fidelity Assembly of Influenza A Virus Promotes Escape from Host Cells

1. Download high-res image (297KB)
2. Download full-size image

     

口蹄疫病毒诱导宿主细胞凋亡的研究

本文报道了口蹄疫病毒（Foot-and-Mouth Disease Virus,FMDV）在体外诱导PK－15细胞凋亡的研究结果,采用Hoechst33258荧光探针、DNA凝胶电泳、脱氧核糖核酸转移酶介导的制品末端标记（TUNEL）技术均检测到了典型的细胞凋亡,结果显示：使用感染性滴度为4.8lgTCID50/mL的口蹄疫病毒感染PK－15细胞,在培养32h后,荧光探针检测呈现典型的凋亡细胞核固缩和梅花状碎裂核,并伴随有凋亡小体出现,调亡率约为20％;DNA凝胶电泳显示ladder梯带;末端标记检测到强绿色荧光标记物结合于凋亡细胞核上。研究结果提示：口蹄疫病毒可以在体外诱导宿主细胞凋亡,细胞凋亡是其致细胞病变死亡的重要途径之一。     

A型流感病毒NS1蛋白与宿主的相互作用

A型流感病毒非结构蛋白1(nonstructural protein l, NS1)全长约为230个氨基酸,主要包括两个功能结构域,即 N-末端的RNA结合结构域和C-末端的效应结构域.NS1是一个多功能病毒蛋白,它不仅影响着该病毒其他基因的表达,更能通过与宿主细胞多种因子的相互作用干预宿主细胞的正常功能,抵抗宿主的抗病毒系统.因此, NS1被认为是A型流感病毒的一个重要毒力因子.本文综述了 NS1蛋白与宿主相互作用的最新研究进展,为进一步揭示NS1 蛋白的功能提供了参考.     

On Hepatitis C Virus Evolution: The Interaction between Virus and Host towards Treatment Outcome

Background

Hepatitis C is a disease spread throughout the world. Hepatitis C virus (HCV), the etiological agent of this disease, is a single-stranded positive RNA virus. Its genome encodes a single precursor protein that yields ten proteins after processing. NS5A, one of the non-structural viral proteins, is most associated with interferon-based therapy response, the approved treatment for hepatitis C in Brazil. HCV has a high mutation rate and therefore high variability, which may be important for evading the immune system and response to therapy. The aim of this study was to analyze the evolution of NS5A quasispecies before, during, and after treatment in patients infected with HCV genotype 3a who presented different therapy responses.

Methods

Viral RNA was extracted, cDNA was synthesized, the NS5A region was amplified and cloned, and 15 clones from each time-point were sequenced. The sequences were analyzed for evolutionary history, genetic diversity and selection.

Results

This analysis shows that the viral population that persists after treatment for most non-responder patients is present in before-treatment samples, suggesting it is adapted to evade treatment. In contrast, the population found in before treatment samples from most end-of-treatment responder patients either are selected out or appears in low frequency after relapse, therefore changing the population structure. The exceptions illustrate the uniqueness of the evolutionary process, and therefore the treatment resistance process, in each patient.

Conclusion

Although evolutionary behavior throughout treatment showed that each patient presented different population dynamics unrelated to therapy outcome, it seems that the viral population from non-responders that resists the treatment already had strains that could evade therapy before it started.