Biological properties of a type C virus isolated from a human X mouse hybrid cell line.

The biological properties of the HMV-1 virus, spontaneously released from a human X C57BL/6 mouse hybrid cell line, were similar to those of RadLV, the prototype B-tropic virus of C57BL/6 mice. Both viruses replicated on B-type mouse cells and in the wild mouse cell line SC-1. The plaque-forming abilities of the two viruses were relatively low, but gradually increased after passage in new host cells. Both viruses were neutralized by AKR antisera but not by FMR antisera. HMV-1 virus could rescue the defective sarcoma genome from S+H- mouse cells. The pseudotype sarcoma virus so produced was deficient in "helper virus" activity. Newborn mice inoculated with HMV-1 virus remained tumor-free over a 1-yr observation period.     

Biophysical analysis of HTLV-1 particles reveals novel insights into particle morphology and Gag stoichiometry

Background

The human immunodeficiency virus type 1 (HIV-1) Vpu protein degrades CD4 and counteracts a restriction factor termed tetherin (CD317; Bst-2) to enhance virion release. It has been suggested that both functions can be genetically separated by mutation of a serine residue at position 52. However, recent data suggest that the S52 phosphorylation site is also important for the ability of Vpu to counteract tetherin. To clarify this issue, we performed a comprehensive analysis of HIV-1 with a mutated casein kinase-II phosphorylation site in Vpu in various cell lines, primary blood lymphocytes (PBL), monocyte-derived macrophages (MDM) and ex vivo human lymphoid tissue (HLT).

Results

We show that mutation of serine 52 to alanine (S52A) entirely disrupts Vpu-mediated degradation of CD4 and strongly impairs its ability to antagonize tetherin. Furthermore, casein-kinase II inhibitors blocked the ability of Vpu to degrade tetherin. Overall, Vpu S52A could only overcome low levels of tetherin, and its activity decreased in a manner dependent on the amount of transiently or endogenously expressed tetherin. As a consequence, the S52A Vpu mutant virus was unable to replicate in macrophages, which express high levels of this restriction factor. In contrast, HIV-1 Vpu S52A caused CD4+ T-cell depletion and spread efficiently in ex vivo human lymphoid tissue and PBL, most likely because these cells express comparably low levels of tetherin.

Conclusion

Our data explain why the effect of the S52A mutation in Vpu on virus release is cell-type dependent and suggest that a reduced ability of Vpu to counteract tetherin impairs HIV-1 replication in macrophages, but not in tissue CD4+ T cells.     

Iquitos virus: a novel reassortant Orthobunyavirus associated with human illness in Peru

Oropouche (ORO) virus, a member of the Simbu serogroup, is one of the few human pathogens in the Orthobunyavirus genus in the family Bunyaviridae. Genetic analyses of ORO-like strains from Iquitos, Peru, identified a novel reassortant containing the S and L segments of ORO virus and the M segment of a novel Simbu serogroup virus. This new pathogen, which we named Iquitos (IQT) virus, was first isolated during 1999 from a febrile patient in Iquitos, an Amazonian city in Peru. Subsequently, the virus was identified as the cause of outbreaks of "Oropouche fever" during 2005 and 2006 in Iquitos. In addition to the identification of 17 isolates of IQT virus between 1999 and 2006, surveys for neutralizing antibody among Iquitos residents revealed prevalence rates of 14.9% for ORO virus and 15.4% for IQT virus. Limited studies indicate that prior infection with ORO virus does not seem to protect against disease caused with the IQT virus infection. Identification of a new Orthobunyavirus human pathogen in the Amazon region of Peru highlights the need for strengthening surveillance activities and laboratory capabilities, and investigating the emergence of new pathogens in tropical regions of South America.     

Characterization of Ribonucleic Acid from Visna Virus

A single-stranded ribonucleic acid(s) has been isolated from purified virions of visna virus. It consists of two major components, namely 63S and "4S," under the conditions employed for ribonucleic acid (RNA) extraction. The 63S component can be converted to subunits by heat and dimethylsulfoxide treatments. Analyses by base composition indicate that the "4S" RNA isolated from visna virus is not a random breakdown product of the 63S component as a result of extraction, nor is it randomly derived from cellular RNA.     

Cleavage of a Neuroinvasive Human Respiratory Virus Spike Glycoprotein by Proprotein Convertases Modulates Neurovirulence and Virus Spread within the Central Nervous System

Human coronaviruses (HCoV) are respiratory pathogens that may be associated with the development of neurological diseases, in view of their neuroinvasive and neurotropic properties. The viral spike (S) glycoprotein is a major virulence factor for several coronavirus species, including the OC43 strain of HCoV (HCoV-OC43). In an attempt to study the role of this protein in virus spread within the central nervous system (CNS) and neurovirulence, as well as to identify amino acid residues important for such functions, we compared the sequence of the S gene found in the laboratory reference strain HCoV-OC43 ATCC VR-759 to S sequences of viruses detected in clinical isolates from the human respiratory tract. We identified one predominant mutation at amino acid 758 (from RRSR↓ G ₇₅₈ to RRSR↓R ₇₅₈), which introduces a putative furin-like cleavage (↓) site. Using a molecular cDNA infectious clone to generate a corresponding recombinant virus, we show for the first time that such point mutation in the HCoV-OC43 S glycoprotein creates a functional cleavage site between the S1 and S2 portions of the S protein. While the corresponding recombinant virus retained its neuroinvasive properties, this mutation led to decreased neurovirulence while potentially modifying the mode of virus spread, likely leading to a limited dissemination within the CNS. Taken together, these results are consistent with the adaptation of HCoV-OC43 to the CNS environment, resulting from the selection of quasi-species harboring mutations that lead to amino acid changes in viral genes, like the S gene in HCoV-OC43, which may contribute to a more efficient establishment of a less pathogenic but persistent CNS infection. This adaptative mechanism could potentially be associated with human encephalitis or other neurological degenerative pathologies.     

Ani immunomodulator Hepon inhibits hepatitis C virus replication in human cell cultures in vitro]

Antiviral activity of immunomodulator "Hepon" was evaluated in human cells culture infected with hepatitis C virus. "Hepon" presence protected human cells SW-13 from cytopathogenic effect of hepatitis C virus. Maximum antiviral effect was demonstrated by "Hepon" at concentration 1 mcg/mL. Control antiviral agent reaferon (interferon alfa-2a) was more potent as vitality protecting agent in the case of SW-13 human cells culture. "Hepon" activity is based on changes of cytokins and interferons spectrum so this immunomodulator is expected to be effective against different viruses including herpes virus and encephalocarditis virus.     

Phylogenetic evidence against evolutionary stasis and natural abiotic reservoirs of influenza A virus

Zhang et al. (G. Zhang, D. Shoham, D. Gilichinsky, S. Davydov, J. D. Castello, and S. O. Rogers, J. Virol. 80:12229-12235, 2006) have claimed to have recovered influenza A virus RNA from Siberian lake ice, postulating that ice might represent an important abiotic reservoir for the persistence and reemergence of this medically important pathogen. A rigorous phylogenetic analysis of these influenza A virus hemagglutinin gene sequences, however, indicates that they originated from a laboratory reference strain derived from the earliest human influenza A virus isolate, WS/33. Contrary to Zhang et al.'s assertions that the Siberian "ice viruses" are most closely related either to avian influenza virus or to human influenza virus strains from Asia from the 1960s (Zhang et al., J. Virol. 81:2538 [erratum], 2007), they are clearly contaminants from the WS/33 positive control used in their laboratory. There is thus no credible evidence that environmental ice acts as a biologically relevant reservoir for influenza viruses. Several additional cases with findings that seem at odds with the biology of influenza virus, including modern-looking avian influenza virus RNA sequences from an archival goose specimen collected in 1917 (T. G. Fanning, R. D. Slemons, A. H. Reid, T. A. Janczewski, J. Dean, and J. K. Taubenberger, J. Virol. 76:7860-7862, 2002), can also be explained by laboratory contamination or other experimental errors. Many putative examples of evolutionary stasis in influenza A virus appear to be due to laboratory artifacts.     

Speculation on Pathogenesis in Death from Respiratory Syncytial Virus Infection

The lungs of three infants, two with bronchiolitis and one with pneumonia, were examined by fluorescent antibody techniques for the distribution of respiratory syncytial (R.S.) virus, and also for the presence of human globulin. In bronchiolitis the lungs contained little virus, whereas in pneumonia virus was abundant and widespread; and, paradoxically, while in bronchiolitis human globulin had the same scanty distribution as virus it was absent in pneumonia. It is suggested that the essential process in bronchiolitis is a widespread type 1 allergic reaction dependent on a second encounter with R.S. virus antigen, whereas in R.S. virus pneumonia the mucosal necrosis and alveolar and interstitial inflammation are the result of direct virus damage to the lungs. The alternative explanation put forward is that the process may be a type 3 allergic reaction.     

Identification of major histocompatibility complex class I C molecule as an attachment factor that facilitates coronavirus HKU1 spike-mediated infection

Human coronavirus HKU1 (HCoV-HKU1) is a recently discovered human coronavirus associated with respiratory tract infections worldwide. In this study, we have identified the major histocompatibility complex class I C molecule (HLA-C) as an attachment factor in facilitating HCoV-HKU1 spike (S)-mediated infection. HCoV-HKU1 S pseudotyped virus was assembled using a human immunodeficiency virus type 1-derived reporter virus harboring the human codon-optimized spike of HCoV-HKU1. We identified human alveolar epithelial A549 cells as the most susceptible cell line among those tested to infection by HCoV-HKU1 S pseudotypes. A549 cells were shown to bind purified soluble HCoV-HKU1 S(1-600) glycopeptide. To search for the functional receptor for HCoV-HKU1, an A549 cDNA expression library was constructed and transduced into the nonpermissive, baby hamster kidney cells line BHK-21. Transduced cells that bind soluble HCoV-HKU1 S(1-600) glycoprotein with C-terminal FLAG were sorted. Sequencing of two independent clones revealed cDNA inserts encoding HLA-C. Inhibition of HLA-C expression or function by RNAi silencing and anti-HLA-C antibody decreased HCoV-HKU1 S pseudotyped virus infection of A549 cells by 62 to 65%, whereas pretreatment of cells with neuraminidase decreased such infection by only 13%. When HLA-C was constitutively expressed in another nonpermissive cell line, NIH-3T3, quantitative PCR showed that the binding of HCoV-HKU1 S pseudotyped virus to cell surfaces was increased by 200-fold, but the cells remained nonsusceptible to HCoV-HKU1 S pseudotyped virus infection. Our data suggest that HLA-C is involved in the attachment of HCoV-HKU1 to A549 cells and is a potential candidate to facilitate cell entry. However, other unknown surface proteins on A549 cells may be concomitantly utilized by S glycoprotein of HCoV-HKU1 during viral entry. Further studies are required to elucidate other putative receptors or coreceptors for HCoV-HKU1 and the mechanism of HCoV-HKU1 S-mediated cell entry.     

Sedimentation characteristics of virion RNA of Machupo virus reproducing in the presence of actinomycin D

Actinomycin D treatment (0.005-05 g/ml) of Vero and BHK-21 cells infected with Machupo virus suppressed the synthesis of ribosomal RNAs but did not affect the production of infectious Machupo virus. Virion RNAs contained 3 high molecular weight RNA species: 28-31 S, 22-24 S and 18 S. In the presence of actinomycin D [3H]-uridine incorporated only in 30-31 S and 22-24 S RNA species. The data are supported by previous results which show that Machupo virus genome contains two RNA species: "large" (30-31 S) and "small" (22-24 S).     

Constitutive Expression of Interferon-Induced Human MxA Protein in Transgenic Tobacco Plants Does not Confer Resistance to a Variety of RNA Viruses

MxA is a key component in the interferon-induced antiviral defense in humans. After viral infections, MxA is rapidly induced and accumulates in the cytoplasm. The multiplication of many RNA viruses,including all bunyaviruses tested so far, is inhibited by MxA. These findings prompted us to express MxA in plants in an attempt to create resistance to tospoviruses. Here, we report the generation of transgenic tobacco plants that constitutively express MxA under the control of the 35S cauliflower mosaic virus promotor. Northern and western blot analysis confirmed the expression of MxA in several transgenic plant lines. MxA expression had no obvious detrimental effects on plant growth and fertility. However, challenge experiments with tomato spotted wilt virus, tomato chlorotic spot virus, and groundnut ringspot virus revealed no increased resistance of MxA-transgenic tobacco plants to tospovirus infections. Neither was the multiplicationof tobacco mosaic virus, cucumber mosaic virus and potato virus Y inhibited in MxA-transgenic plants. The results indicate that the expression of human MxA alone does not enhance virus resistance in planta.     

Sphingosine 1-Phosphate-Metabolizing Enzymes Control Influenza Virus Propagation and Viral Cytopathogenicity

Sphingosine 1-phosphate (S1P)-metabolizing enzymes regulate the level of sphingolipids and have important biological functions. However, the effects of S1P-metabolizing enzymes on host defense against invading viruses remain unknown. In this study, we investigated the role of S1P-metabolizing enzymes in modulating cellular responses to influenza virus infection. Overexpression of S1P lyase (SPL), which induces the degradation of S1P, interfered with the amplification of infectious influenza virus. Accordingly, SPL-overexpressing cells were much more resistant than control cells to the cytopathic effects caused by influenza virus infection. SPL-mediated inhibition of virus-induced cell death was supported by impairment of the upregulation of the proapoptotic protein Bax, a critical factor for influenza virus cytopathogenicity. Importantly, influenza virus infection of SPL-overexpressing cells induced rapid activation of extracellular signal-regulated kinase (ERK) and STAT1 but not of p38 mitogen-activated protein kinase (MAPK), Akt, or c-Jun N-terminal kinase (JNK). Blockade of STAT1 expression or inhibition of Janus kinase (JAK) activity elevated the level of influenza virus replication in the cells, indicating that SPL protects cells from influenza virus via the activation of JAK/STAT signaling. In contrast to that of SPL, the overexpression of S1P-producing sphingosine kinase 1 heightened the cells'' susceptibility to influenza virus infection, an effect that was reversed by the inhibition of its kinase activity, representing opposed enzymatic activity. These findings indicate that the modulation of S1P-metabolizing enzymes is crucial for controlling the host defense against infection with influenza virus. Thus, S1P-metabolizing enzymes are novel potential targets for the treatment of diseases caused by influenza virus infection.Influenza virus continues to threaten humans and remains a major worldwide health concern. Influenza virus causes an average of 36,000 deaths and 200,000 hospitalizations annually in the United States (50), imposing a significant economic burden (33). Further, there is fear of the recurrence of a devastating influenza pandemic similar to the Spanish influenza pandemic in 1918/1919, which killed as estimated 40 to 50 million people worldwide (34). Indeed, on 11 June 2009, the World Health Organization (WHO) declared the spread of the 2009 influenza A (H1N1) virus (initially known as swine flu virus) a global influenza pandemic (14, 45, 51). In addition, outbreaks of avian H5N1 influenza elevated vigilance against the occurrence of an influenza pandemic (4). A substantial number of circulating seasonal influenza viruses, as well as the avian H5N1 influenza virus with pandemic potential, were found to be resistant to antiviral drugs (10). Thus, identifying new therapeutic targets and understanding the mechanisms of host-virus interactions are important biomedical goals.Sphingolipids are bioactive lipid mediators characterized by the presence of a serine head group with one or two fatty acid tails (7, 44). One of the sphingolipids, sphingosine, and its downstream product sphingosine 1-phosphate (S1P), have emerged as the modulators of multiple cellular processes, such as cell growth, survival, differentiation, and migration, and have therapeutic potential. For instance, a sphingosine analog, FTY720, is a promising biomedical drug candidate that is currently being tested in phase III clinical trials for the treatment of multiple sclerosis (20). S1P, which is generated inside cells, can trigger intracellular signaling or is secreted to act as an exogenous lipid mediator stimulating S1P receptor-mediated signaling (44, 47).The level of S1P is tightly regulated by the S1P-metabolizing enzymes sphingosine kinase (SK) and S1P lyase (SPL). Its synthesis from sphingosine is catalyzed by SK, while SPL catalyzes the degradation of S1P to phosphoethanolamine and hexadecanal (46). These S1P-metabolizing enzymes were revealed to modulate diverse cellular stresses induced by anticancer drugs (30, 31), DNA damage (39), or serum deprivation (38, 43). Cells overexpressing SK1 displayed increased resistance to anticancer drugs such as cisplatin, carboplatin, and doxorubicin (30), whereas cells overexpressing SPL were more sensitive to drug-mediated cell death (31).Recently, the sphingosine analog AAL-R was shown to display immunomodulatory activity to alleviate influenza virus-induced immune pathology (27, 28). The phosphorylated analog acted directly on S1P receptors to regulate the expression of inflammatory cytokines, although it did not significantly alter influenza virus propagation (28). However, the role of intracellular S1P-metabolizing enzymes in host defensive mechanisms against influenza virus infection has not been studied.Here, we now show the contribution of the S1P-metabolizing enzymes SPL and SK1 to cellular responses to influenza virus infection. Overexpression of SPL interfered with influenza virus amplification and virus-induced cell death, with the early activation of STAT1 and extracellular signal-regulated kinase (ERK) molecules. Treatment with inhibitors blocking STAT1 expression or Janus kinase 1 (JAK1) activation increased influenza virus replication preferentially in SPL-overexpressing cells, demonstrating the importance of JAK/STAT signaling for SPL-mediated host defense. The suppression of influenza virus-induced cellular apoptosis by SPL was supported by the diminished expression of both the proapoptotic protein Bax and the cleaved product of poly(ADP-ribose) polymerase (PARP). In contrast, the overexpression of SK1 made cells more permissive to influenza virus infection, which was reversed by the inhibition of its kinase activity. Collectively, our results demonstrate that S1P-metabolizing enzymes regulate influenza virus propagation and represent novel therapeutic targets.     

Selective synthesis and secretion of particles composed of the hepatitis B virus middle surface protein directed by a recombinant vaccinia virus: induction of antibodies to pre-S and S epitopes.

Selective synthesis in mammalian cells of the hepatitis B virus middle surface (MS) protein, which is 55 amino acids longer than the major surface (S) protein, was achieved by using a recombinant vaccinia virus. The 33-kilodalton MS polypeptide was glycosylated and secreted as particles that resembled human hepatitis B surface antigen as well as particles composed solely of S protein with regard to antigenicity, buoyant density, size, and electron micrographic appearance. The MS particles differed from S particles, however, by binding to polymerized human albumin and inducing antibodies that reacted with a pre-S peptide and inhibited the binding of human plasma-derived hepatitis B surface antigen to polymerized human albumin.     

Distinct induction patterns and functions of two closely related interferon-inducible human genes, ISG54 and ISG56

Human P54 and P56 proteins are tetratricopeptide proteins that are encoded by two closely related genes, ISG54 and ISG56. These genes are induced strongly but transiently when cells are treated with interferons or double-stranded RNA or infected with a variety of viruses. We observed that, although double-stranded RNA or Sendai virus infection induced the two genes with similar kinetics, their induction kinetics in response to interferon-beta were quite different. The induction kinetics by virus infection were also different between two cell lines. Functionally the two proteins were similar. Like P56, P54 bound to the translation initiation factor eIF3 and inhibited translation. However, unlike P56, P54 bound to both the "e" and the "c" subunits of eIF3. Consequently, P54 inhibited two functions of eIF3. Like P56, it inhibited the ability of eIF3 to stabilize the eIF2 x GTP x Met-tRNA(i) ternary complex. But in addition, it also inhibited the formation of the 48 S pre-initiation complex between the 40 S ribosomal subunit and the 20 S complex composed of eIF3, ternary complex, eIF4F, and mRNA. Thus, although similar in structure, the human P54 and P56 proteins are induced differently and function differently.     

Zur „Immunität“ der Sorte Saco gegenüber dem S-Virus der Kartoffel

Zusammenfassung Im Rahmen eines 1962 begonnenen Programmes zur Resistenzzüchtung bei S-Virus wurde auch die Immunität der Sorte Saco gegenüber dem S-Virus nochmals überprüft. Diese Überprüfung erfolgte sowohl durch Abreibung mit S-virushaltigem Preßsaft wie auch durch Pfropfung mit einem S-besetzten Reis einer anfälligen Sorte. Bei der Abreibung des virushaltigen Preßsaftes kam es nicht zu einer faßbaren Virusvermehrung, jedoch erwiesen sich im erstjährigen Nachbau 2 von 16 Pflanzen als infiziert, im zweitjährigen Nachbau konnte in diesen Pflanzen kein S-Virus mehr nachgewiesen werden. Infektion über Pfropfung ergab bereits im Infektionsjahr bei 4 von 8 Pflanzen eine S-Virusvermehrung. Im ersten Nachbau erwiesen sich 7 von 8 Pflanzen als infiziert. Der zweitjährige Nachbau brachte ebenfalls einen Rückgang in der Konzentration des S-Virus und in der Zahl der mit S-Virus verseuchten Pflanzen. In Übereinstimmung mit Bagnall (1965) zeigen die durchgeführten Untersuchungen, daß die Sorte Saco keine Immunität, sondern nur einen hohen Grad von Resistenz gegenüber dem S-Virus besitzt.

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On the immunity of the variety Saco to potato virus S.

Summary During a breeding program for resistance to virus S, started in 1962, the immunity of the variety Saco to virus S was checked once again. The test was carried out both by rubbing in a sap containing virus S and by grafting a virus-S-infected scion of a susceptible variety. After rubbing in the virus containing sap no detectable virus multiplication could be observed, however in the first year's offspring 2 of 16 plants were infected, in the offspring of the second year no more virus S could be detected. Infection by grafting showed multiplication of virus S in 4 of 8 plants already in the year of infection. In the first generation 7 of 8 plants proved infected. The offspring of the second year also showed a reduction of virus S concentration and in number of virus S infected plants. In accordance with Bagnall (1965) the performed trials show that the variety Saco possesses no immunity but a high degree of resistance to virus S.

     

Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α1-Antitrypsin Variants

Background

Proteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease (S1P) is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and, hence, essential for infectivity and virus spread. Therefore, we tested in this study the concept of using S1P as a target to block efficient virus replication.

Methodology/Principal Finding

We demonstrate that stable cell lines inducibly expressing S1P-adapted α₁-antitrypsin variants inhibit the proteolytic maturation of GP-C. Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of α₁-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells. Moreover, S1P-specific α₁-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus. Inhibition of viral replication correlated with the ability of the different α₁-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor.

Conclusions/Significance

Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies.     

A Detailed Phylogenetic Analysis of FIV in the United States

Background

Feline immunodeficiency virus (FIV) is a lentivirus associated with AIDS-like illnesses in cats and has been used as a model for the study of human immunodeficiency virus (HIV). A feature of HIV and FIV infection is the continually increasing divergence among viral isolates between different individuals, as well as within the same individuals.

Methodology/Principal Findings

The goal of this study was to determine the phylogenetic patterns of viral isolates obtained within the United States (U.S.) by focusing on the variable, V3-V4, region of the FIV envelope gene.

Conclusions/Significance

Data indicate that FIV, from within the U.S., localize to four viral clades, A, B, C, and F. Also shown is the geographic isolation of strains where clade A and clade B are found predominately on the west coast; however, clade B is also found throughout the U.S. and represents the predominant clade. This study presents a complete and conclusive analysis of FIV isolates from within the U.S. and may be used as the essential basis for the development of an effective multi-clade vaccine.