ABSTRACT
We studied the kinetics of localisation of matrix (M) protein of Human respiratory syncytial virus (RSV) in infected cells. M protein was detected in the nucleus early in infection, by confocal microscopy and by immunoblotting of nuclear fractions. We next tested the possibility that M protein may be involved in inhibition of host cell transcription. Nuclear extracts from RSV infected cells had less transcriptional activity in vitro when compared to nuclear extracts from mock infected cells. In addition, nuclear extracts from RSV infected cells inhibited the transcriptional activity of nuclear extracts from mock infected cells, suggesting that an inhibitory activity was transferred with nuclear extracts from RSV infected cells. Our data suggest that M protein may play a role early in the infection by inhibiting host cell transcription.
Subject(s)
Cell Nucleus/virology , Gene Expression Regulation, Viral , Respiratory Syncytial Virus, Human/genetics , Transcription, Genetic/genetics , Viral Matrix Proteins/metabolism , Binding Sites , Carcinoma, Hepatocellular , Humans , Kinetics , Liver Neoplasms , Respiratory Syncytial Virus, Human/pathogenicity , Tumor Cells, Cultured , Viral Proteins/geneticsABSTRACT
Expression of the chemokine receptor CXCR4 on human haemopoietic stem cells (HSC) may play a crucial role in localising these cells to the bone marrow. To evaluate whether CXCR4 expression is clinically relevant we have enumerated CXCR4-positive HSC used for allogeneic transplantation and sought any relationship with the rate of subsequent haemopoietic reconstitution. CD34-positive progenitor cells were isolated from peripheral blood stem cell (PBSC) collections from 16 normal donors. The proportion of cells co-expressing CXCR4 was enumerated and the times to recipient haemopoietic reconstitution measured. The median frequency of CD34-positive cells co-expressing CXCR4 was 41% (range 16% to 76%) and the median number of CXCR4 CD34 double-positive cells infused at transplantation was 2.5 x 10(6) cells/kg (range 0.8-10.3). Patients receiving >2.5 x 10(6) CXCR4 CD34 double-positive cells/kg demonstrated a significant shortening of time to platelet engraftment compared to the recipients of the lower cell doses (10 days vs 14.5 days, respectively, P = 0.02) with all but one of the recipients of the higher cell doses achieving platelet engraftment by day 11. Co-expression of CXCR4 on CD34-positive progenitor cells may be an important determinant of post-transplant engraftment and in our hands transplantation of a minimum of 2.5 x 10(6) CXCR4 CD34 double-positive cells/kg ensured rapid post-transplant platelet recovery.
Subject(s)
Bone Marrow Cells/drug effects , Bone Marrow Cells/physiology , Bone Marrow/physiology , Granulocyte Colony-Stimulating Factor/therapeutic use , Hematopoietic Stem Cell Mobilization/methods , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/physiology , Tissue Donors , Adult , Hematopoietic Stem Cells/cytology , Humans , Leukocyte Count/methods , Transplantation, HomologousABSTRACT
The capsid protein of Australian feline calicivirus (FCV) isolates is demonstrably different from the prototype strain F9. Five Australian isolates of FCV, dating from 1970 to 1989, were analysed by western blotting and immunoprecipitation. Varying reactivity to a panel of F9 specific monoclonal antibodies (MAbs) was observed. DNA sequencing of RT-PCR generated clones supported the observation of variation between capsid proteins. Predicted amino acid sequences varied by 11 to 17.5% across the whole capsid when compared to the published F9 sequence. Differences in amino acid sequence were most apparent in previously described hypervariable regions (C and E). Within hypervariable region E differences of 22 to 34% were observed compared to F9. The observed lack of reactivity to F9 MAbs correlated with amino acid changes within previously characterized binding sites within region E.
Subject(s)
Caliciviridae Infections/veterinary , Calicivirus, Feline/classification , Capsid/chemistry , Cat Diseases/virology , Genetic Variation , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Australia , Blotting, Western/veterinary , Caliciviridae Infections/virology , Calicivirus, Feline/chemistry , Calicivirus, Feline/genetics , Calicivirus, Feline/immunology , Capsid/genetics , Capsid/immunology , Cats , Consensus Sequence , DNA Primers/chemistry , DNA, Viral/chemistry , Molecular Sequence Data , Phylogeny , Precipitin Tests/veterinary , RNA, Viral/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
We have determined the first complete genome sequence and capsid gene sequences of feline calicivirus (FCV) isolates from the UK and Australia. These were compared with other previously published sequences. The viruses used in the comparisons were isolated between 1957 and 1995 from various geographical locations and obtained from cats showing a range of clinical signs. Despite these diverse origins, comparisons between all strains showed a similar degree of sequence variation within both ORF1 (non-structural polyprotein) and ORF2 (major capsid protein) (amino acid distances of 7.7-13.0% and 8.8-18.6%, respectively). In contrast, ORF3 (putative minor structural protein) sequences indicated a more heterogenous distribution of FCV relatedness (amino acid distances of 1.9-17.9%). Phylogenetic analysis suggested that, unlike some other caliciviruses, FCV isolates within the current data set fall into one diverse genogroup. Within this group, there was an overall lack of geographic or temporal clustering which may be related to the epidemiology of FCV infection in cats. Analysis of regions of variability in the genome has shown that, as well as the previously identified variable regions in ORF2, similar domains exist within ORFs 1 and 3 also, although to a lesser extent. In ORF1, these variable domains largely fall between the putative non-structural protein functional domains.
