Isolation of infectious salmon anemia virus (ISAV) from Atlantic salmon in New Brunswick, Canada.
(17/5443)
Infectious salmon anemia virus (ISAV) was isolated at a marine grow-out site in New Brunswick, Canada, from Atlantic salmon Salmo salar which experienced mortalities due to hemorrhagic kidney syndrome (HKS). Of 20 fish sampled in this study, 14 showed histologically various degrees of interstitial hemorrhaging, tubular epithelial degeneration and necrosis, and tubular casts in the posterior kidney, typical of HKS. Posterior kidney and spleen homogenates produced a cytopathic effect on chinook salmon embryo (CHSE-214) cells 10 to 14 d after inoculation. Pleomorphic virus particles in the size range 80 to 120 nm were seen by electron microscopy. The virus was confirmed as ISAV using reverse transcriptase-polymerase chain reaction (RT-PCR). This is a systematic diagnostic study of the isolation of ISAV on the North American continent and the first description of the growth of ISAV on the CHSE-214 cell line. (+info)
Human DNA topoisomerase I-mediated cleavage and recombination of duck hepatitis B virus DNA in vitro.
(18/5443)
In this study, we report that eukaryotic topoisomerase I (top1) can linearize the open circular DNA of duck hepatitis B virus (DHBV). Using synthetic oligonucleotides mimicking the three-strand flap DR1 region of the DHBV genome, we found that top1 cleaves the DNA plus strand in a suicidal manner, which mimics the linearization of the virion DNA. We also report that top1 can cleave the DNA minus strand at specific sites and can linearize the minus strand via a non-homologous recombination reaction. These results are consistent with the possibility that top1 can act as a DNA endo-nuclease and strand transferase and play a role in the circularization, linearization and possibly integration of viral replication intermediates. (+info)
Analysis of human lymphotropic T-cell virus type II-like particle production by recombinant baculovirus-infected insect cells.
(19/5443)
The molecular processes involved in retrovirus assembly and budding formation remain poorly understood. The gag-pro-pol genes of human lymphotropic T-cell virus type II (HTLV-II) are translated into Gag, Gag-Pro, or Gag-Pro-Pol by frameshift events. In the present study, we investigated the roles of the gag, pro, and pol regions of HTLV-II in viral particle formation using recombinant baculoviruses. In this study we could successfully produce mature HTLV-II viral particles containing core structures using a construct expressing the entire gag-pro-pol region. We also investigated the role of the pol region in particle formation. Deletion of the pol region affects viral particle assembly or release very little, indicating that the gag-pro region is sufficient for viral particle formation and maturation. Expression of the Gag proteins alone or Gag proteins with inactivated viral proteases (Pro) resulted in the formation of viral particles; however, these particles did not contain core structures. These results suggest the intracellular expression of Gag with Pro of HTLV-II is essential for the production of mature virus particles, whereas that of Pol is not. (+info)
Role of rubella virus glycoprotein domains in assembly of virus-like particles.
(20/5443)
Rubella virus is a small enveloped positive-strand RNA virus that assembles on intracellular membranes in a variety of cell types. The virus structural proteins contain all of the information necessary to mediate the assembly of virus-like particles in the Golgi complex. We have recently identified intracellular retention signals within the two viral envelope glycoproteins. E2 contains a Golgi retention signal in its transmembrane domain, whereas a signal for retention in the endoplasmic reticulum has been localized to the transmembrane and cytoplasmic domains of E1 (T. C. Hobman, L. Woodward, and M. G. Farquhar, Mol. Biol. Cell 6:7-20, 1995; T. C. Hobman, H. F. Lemon, and K. Jewell, J. Virol. 71:7670-7680, 1997). In the present study, we have analyzed the role of these retention signals in the assembly of rubella virus-like particles. Deletion or replacement of these domains with analogous regions from other type I membrane glycoproteins resulted in failure of rubella virus-like particles to be secreted from transfected cells. The E1 transmembrane and cytoplasmic domains were not required for targeting of the structural proteins to the Golgi complex and, surprisingly, assembly and budding of virus particles into the lumen of this organelle; however, the resultant particles were not secreted. In contrast, replacement or alteration of the E2 transmembrane or cytoplasmic domain, respectively, abrogated the targeting of the structural proteins to the budding site, and consequently, no virion formation was observed. These results indicate that the transmembrane and cytoplasmic domains of E2 and E1 are required for early and late steps respectively in the viral assembly pathway and that rubella virus morphogenesis is very different from that of the structurally similar alphaviruses. (+info)
Regulation of vaccinia virus morphogenesis: phosphorylation of the A14L and A17L membrane proteins and C-terminal truncation of the A17L protein are dependent on the F10L kinase.
(21/5443)
This study focused on three vaccinia virus-encoded proteins that participate in early steps of virion morphogenesis: the A17L and A14L membrane proteins and the F10L protein kinase. We found that (i) the A17L protein was cleaved at or near an AGX consensus motif at amino acid 185, thereby removing its acidic C terminus; (ii) the nontruncated form was associated with immature virions, but only the C-terminal truncated protein was present in mature virions; (iii) the nontruncated form of the A17L protein was phosphorylated on serine, threonine, and tyrosine residues, whereas the truncated form was unphosphorylated; (iv) nontruncated and truncated forms of the A17L protein existed in a complex with the A14L membrane protein; (v) C-terminal cleavage of the A17L protein and phosphorylation of the A17L and A14L proteins failed to occur in cells infected with a F10L kinase mutant at the nonpermissive temperature; and (vi) the F10L kinase was the only viral late protein that was necessary for phosphorylation of the A17L protein, whereas additional proteins were needed for C-terminal cleavage. We suggest that phosphorylation of the A17L and A14L proteins is mediated by the F10L kinase and is required to form the membranes associated with immature virions. Removal of phosphates and the A17L acidic C-terminal peptide occur during the transition to mature virions. (+info)
Attenuated vesicular stomatitis viruses as vaccine vectors.
(22/5443)
We showed previously that a single intranasal vaccination of mice with a recombinant vesicular stomatitis virus (VSV) expressing an influenza virus hemagglutinin (HA) protein provided complete protection from lethal challenge with influenza virus (A. Roberts, E. Kretzschmar, A. S. Perkins, J. Forman, R. Price, L. Buonocore, Y. Kawaoka, and J. K. Rose, J. Virol. 72:4704-4711, 1998). Because some pathogenesis was associated with the vector itself, in the present study we generated new VSV vectors expressing HA which are completely attenuated for pathogenesis in the mouse model. The first vector has a truncation of the cytoplasmic domain of the VSV G protein and expresses influenza virus HA (CT1-HA). This nonpathogenic vector provides complete protection from lethal influenza virus challenge after intranasal administration. A second vector with VSV G deleted and expressing HA (DeltaG-HA) is also protective and nonpathogenic and has the advantage of not inducing neutralizing antibodies to the vector itself. (+info)
Modulation of phosphate uptake and amphotropic murine leukemia virus entry by posttranslational modifications of PIT-2.
(23/5443)
PIT-2 is a type III sodium phosphate cotransporter and the receptor for amphotropic murine leukemia viruses. We have investigated the expression and the functions of a tagged version of PIT-2 in CHO cells. PIT-2 remained equally abundant at the cell surface within 6 h following variation of the phosphate supply. In contrast, the efficiency of phosphate uptake and retrovirus entry was inversely related to the extracellular phosphate concentration, indicating that PIT-2 activities are modulated by posttranslational modifications of cell surface molecules induced by phosphate. Conformational changes of PIT-2 contribute to both activities, as shown by the inhibitory effect of sulfhydryl reagents known as inhibitors of type II cotransporters. A physical association of PIT-2 with actin was demonstrated. Modifications of the actin network were induced by variations of the concentrations of extracellular phosphate, cytochalasin D, or lysophosphatidic acid. They revealed that the formation of actin stress fibers determines the cell surface distribution of PIT-2, the internalization of the receptor in response to virus binding, and the capacity to process retrovirus entry. Thus, the presence of PIT-2 at the cell surface is not sufficient to ensure phosphate transport and susceptibility to amphotropic retrovirus infection. Further activation of cell surface PIT-2 molecules is required for these functions. (+info)
Identification of a cell surface protein from Crandell feline kidney cells that specifically binds Aleutian mink disease parvovirus.
(24/5443)
Aleutian mink disease parvovirus (ADV) is the etiological agent of Aleutian disease of mink. The acute disease caused by ADV consists of permissive infection of alveolar type II cells that results in interstitial pneumonitis. The permissive infection is experimentally modeled in vitro by infecting Crandell feline kidney (CrFK) cells with a tissue culture-adapted isolate of ADV, ADV-G. ADV-G VP2 empty virions expressed in a recombinant baculovirus system were analyzed for the ability to bind to the surface of CrFK cells. Radiolabeled VP2 virions bound CrFK cells specifically, while they did not bind either Mus dunni or Spodoptera frugiperda cells, cells which are resistant to ADV infection. The binding to CrFK cells was competitively inhibited by VP2 virions but not by virions of cowpea chlorotic mottle virus (CCMV), another unenveloped virus similar in size to ADV. Furthermore, preincubation of CrFK cells with the VP2 virions blocked infection by ADV-G. The VP2 virions were used in a virus overlay protein binding assay to identify a single protein of approximately 67 kDa, named ABP (for ADV binding protein), that demonstrates specific binding of VP2 virions. Exogenously added VP2 virions were able to competitively inhibit the binding of labeled VP2 virions to ABP, while CCMV virions had no effect. Polyclonal antibodies raised against ABP reacted with ABP on the outer surface of CrFK cells and blocked infection of CrFK cells by ADV-G. In addition, VP2 virion attachment to CrFK cells was blocked when the VP2 virions were preincubated with partially purified ABP. Taken together, these results indicate that ABP is a cellular receptor for ADV. (+info)