- Volume 74, Issue 4, 1993

We have previously demonstrated antibody-dependent enhancement of feline infectious peritonitis virus (FIPV) infection of macrophages using both virus-specific antisera and monoclonal antibodies (MAbs) to the spike (S) protein of FIPV. To increase our understanding of this phenomenon, six representative MAbs from a previously documented group of 12 enhancing MAbs were used to identify epitopes that mediate antibody-dependent enhancement of FIPV infectivity. Analysis of the results of kinetics-based competitive ELISA (K-cELISA) among these six enhancing MAbs grouped the epitopes into two clusters. Because transmissible gastroenteritis virus (TGEV) and FIPV are so closely related antigenically, we also conducted K-cELISA experiments between the FIPV MAbs and TGEV S protein-specific MAbs for which the epitopes had previously been mapped to specific sites on the TGEV S protein. Results of these assays indicated that the two FIPV epitope clusters are homologues of the previously defined TGEV S protein sites A and E/F. In addition, two TGEV S protein-specific MAbs also induced antibody-dependent enhancement of FIPV infection of macrophages. This functional cross-reactivity provides further support for the close antigenic relationship between FIPV and TGEV. Our results provide a preliminary localization of several enhancing epitopes within the amino acid sequence of the FIPV S protein.

Cytotoxic T lymphocyte (CTL) responses to vaccinia virus (VV) were studied in human subjects receiving smallpox vaccine by dermal scarification. After in vitro restimulation with VV, peripheral blood mononuclear cells (PBMCs) from three of six vaccinated subjects killed virus-infected target cells. VV-specific, HLA-restricted CTL activity was mediated primarily by CD8+ cells, although low levels of lytic activity by CD4+ cells were observed in some experiments. Two of the three responders had no history of exposure to VV prior to vaccination, whereas all three non-responders were vaccinated against smallpox during childhood. PBMCs from an additional three subjects who had received smallpox vaccine at least 20 years previously were negative for CTL activity. These data suggest that the duration of memory CTL populations against VV in the peripheral blood is limited, and that pre-existing immunity to VV may interfere with boosting of the CTL response upon revaccination.

We have examined intrinsic and external factors that influence human papillomavirus type 16 (HPV-16)-mediated immortalization of oral keratinocytes. The efficiency with which HPV can immortalize human oral keratinocytes was quantified and a considerable difference in the transfection and immortalization competence of the cells was detected. The ability of HPV-16 to immortalize oral cells appeared to be linked to the age of the culture upon transfection. The addition of dexamethasone to the transfected cultures increased the efficiency of immortalization, possibly indicating a role for a critical level of HPV gene expression in initial outgrowth of immortalized colonies. We also document in detail the changes in the oral keratinocyte induced by HPV-16 immortalization. These include alterations associated with crisis and feeder independence as well as basic changes in keratin expression and differentiation.

Bovine papillomavirus type 1 (BPV-1) virions were produced in vitro using vaccinia virus (VV) recombinants expressing the BPV-1 L1 and L2 capsid proteins. Particles morphologically resembling papillomaviruses were observed in the nucleus of cells infected with a VV recombinant for the BPV-1 L1 protein, and greater numbers of similar particles were seen in the nuclei of cells infected with a VV double recombinant for L1 and L2. Virus-like particles (VLPs) assembled in cells infected with the VV double recombinant for BPV-1 L1 and L2, and not those assembled in cells infected with the VV recombinant for BPV-1 L1 alone, were able to package BPV-1 DNA. Transcription of the BPV-1 E1 viral open reading frame was observed after a mouse fibroblast cell line was exposed to VLPs produced using a BPV-1 L1/L2 VV recombinant in a cell line containing episomal BPV-1 DNA. E1 transcription was not observed when the VLPs were pre-incubated with antibodies to the capsid protein of BPV-1. This system should allow an in vitro approach to the definition of the BPV-1 cellular receptor.

The 9∙9 kb monopartite ssRNA genome of parsnip yellow fleck virus (PYFV) encodes a polyprotein from which the functional proteins are assumed to arise by proteolytic cleavage. The 22∙5K, 26K and 31K particle proteins were mapped in the polyprotein by determining their N-terminal amino acid sequences, and were found to begin at amino acid positions 395, 589 and 811, respectively. There could be polypeptide(s) of up to 43K on the N-terminal side of the particle protein sequences. A region within the 26K particle protein has sequence similarity to the VP3 particle protein of picornaviruses. Three other regions in the PYFV polyprotein have sequence similarity to regions thought to have RNA polymerase, NTP-binding and protease functions in the polyproteins of picornaviruses, comoviruses and nepoviruses. Despite these similarities in sequence and in genome organization to viruses in the picorna-like supergroup, PYFV is distinct from all other plant and animal viruses described. This justifies placing it in a separate plant virus genus for which the name ‘sequivirus’ has been proposed.

The complete nucleotide sequence (3514 nucleotides) of RNA segment 2 of rice stripe virus (RSV), the prototype member of tenuivirus group, was determined. In the virus-sense RNA an open reading frame (ORF) is present which encodes a 199 amino acid protein of M r 22762. Another long ORF encoding an 834 amino acid protein with M r 94047 (94K) exists in the virus-complementary RNA. Between these two ORFs, there is a long non-coding intergenic region of 299 nucleotides. The sequence suggests that RNA 2 has an ambisense coding strategy as found for RSV RNAs 3 and 4. The putative 94K protein carries stretches with an amino acid sequence showing weak similarity to parts of the membrane glycoproteins of Punta Toro and Uukuniemi phleboviruses of the family Bunyaviridae, suggesting a possible distinct evolutionary relationship between the animal phleboviruses and the plant tenuiviruses.

Plant pararetroviruses, such as caulimoviruses, and animal retroviruses have in common the presence of a highly conserved arrangement of cysteines and a histidine in the precursor of the capsid protein. The composition of these amino acids resembles a zinc finger element, a structure that is common to a class of eukaryotic proteins that regulate gene expression. The role of the putative zinc finger in the life-cycle of caulimoviruses was investigated by introducing specific mutations in the coat protein coding region of a cloned and infectious form of figwort mosaic virus, a caulimovirus. This mutated viral genome, which no longer encoded the conserved cysteine and histidine residues, was not infectious in plants. Transient expression assays in protoplasts showed that expression of a reporter gene inserted at different places in the genome was not detectably influenced by the coat protein or its putative zinc finger. It appears that the zinc finger-like element of caulimoviruses is not involved in the regulation of gene expression. These observations support a model which predicts a function of the zinc finger in specific recognition and packaging of viral RNA into virions prior to reverse transcription.

A full-length cDNA copy of clover yellow mosaic virus (CYMV) RNA was constructed from two smaller cDNA clones. In vitro transcription of linearized plasmid with T7 RNA polymerase produced genomic-sized RNA. These transcripts caused symptoms typical of CYMV infection when used to inoculate both a systemic host (Vicia faba) and a local lesion host (Gomphrena globosa). Electron microscopy of extracts from individual local lesions revealed virus particles identical to native CYMV. Increasing the length of the poly(A) tail from 23 residues to 80 or to 135 residues increased the infectivity rate from 12% to 17% or to 35% that of native CYMV RNA, respectively.

Cocksfoot mottle sobemovirus supports replication and encapsidation of the viroid-like satellite RNA (sat-RNA) of lucerne transient streak virus (LTSV) in two monocotyledonous species, Triticum aestivum and Dactylis glomerata. Additionally, LTSV sat-RNA replicates effectively in the presence of turnip rosette sobemovirus in Brassica rapa, Raphanus raphanistrum and Sinapsis arvensis, but not in Thlaspi arvense or Nicotiana bigelovii, indicating that host species markedly influence this interaction. Previous reports of the association between LTSV sat-RNA and helper sobemoviruses were limited to dicotyledonous hosts. Our results demonstrate that the biological interaction between these two entities spans divergent dicotyledonous and monocotyledonous species.