- Volume 71, Issue 1, 1990

The close association between human papillomavirus type 16 (HPV-16) and cervical cancer implies some role for the virus in the development of this disease. Recent studies have shown that HPV-16, under the control of strong heterologous promoters, can cooperate with the activated ras oncogene to transform primary baby rat kidney cells. Virus types associated with benign lesions, e.g. HPV-6 and -11, do not function in this system. The discrimination between virus types associated with benign and tumorigenic lesions by this assay implicate it as a useful system for the study of transformation in vitro. The studies reported here investigate the activity of the HP V -16 early gene product E2 in transformation. In the presence of exogenous E2, endogenous viral promoters are stimulated sufficiently to give a high efficiency of transformation in primary epithelial cells. This transactivation by E2 obviates the need for heterologous promoters, and implicates increased viral gene expression as a prerequisite for transformation. The stimulatory effect of E2 appears to be mediated through increased levels of expression of the E7 protein, which has been shown in similar assays to be sufficient to give transformation in cooperation with ras. CAT assays confirm that HPV-16 E2 can transactivate the HPV-16 early promoters. These studies demonstrate some of the elements in a complex series of events likely to be involved in the development of cervical carcinomas.

Nuclear run-on assays carried out in the presence and absence of the RNA polymerase II inhibitor, α-amanitin, were used to determine the exact timing of the switch from inhibitor-sensitive transcription catalysed by host RNA polymerase II, to inhibitor-resistant transcription catalysed by the baculovirus-induced RNA polymerase. These studies revealed that the onset of α-amanitin-resistant transcription is just after 6 h post-infection, simultaneous with the beginning of the late phase of infection. They also showed that transcripts from the p26 gene in the HindIII Q/P region and the p35 gene in the HindIII K/Q region of the viral genome are synthesized by the host RNA polymerase II both early and late in infection. On the other hand, transcripts of the pl0 gene in the HindIII Q/P region and the γ transcripts in the HindIII K region are synthesized by the α-amanitin-resistant, virus-induced RNA polymerase late in infection.

We have mapped a set of transcripts that cross the XhoI site in the HindIII F fragment of the genome of the Autographa californica nuclear polyhedrosis virus. These transcripts overlap at their 5′ ends by about 550 bases and run in opposite directions. We have tentatively identified two open reading frames corresponding to the leftward transcript. The rightward transcript is present from 8 h post-infection (p.i.) to 24 h p.i.; the leftward transcripts are present from 2 h p.i. to 24 h p.i. The early (2 h) transcript is about 2·1 kb in size and its 5′ end maps about 504 bp to the right of the XhoI site. Beginning at about 8 h p.i. a new transcription start site is used, about 80 bp downstream (to the left) of the first. The late (rightward) transcript is about 1·2 kb in size; its 5′ end seems to be heterogeneous and maps about 44 to 60 bp to the left of the XhoI site. Late in infection transcription proceeds in both directions at the same time through the overlapping region of the DNA encoding these transcripts.

Synthetic peptides representing 14 regions of the bovine enterovirus structural proteins were used to raise antibodies in mice. The peptides were predicted using amino acid sequence alignments with the position of antigenic sites on other picornaviruses. Five of the anti-peptide antibodies reacted with the virus in an immunoprecipitation test. Furthermore, each of these anti-peptide antibodies neutralized virus infectivity; those directed against peptides of VP2 and VP3 neutralized to a greater extent than those directed against peptides of VP1. The positions of these epitopes in the viral structural proteins are discussed in relation to corresponding positions in other picornaviruses.

Two monoclonal antibodies (MAbs) were obtained from BALB/c mice immunized with cowpox virus (CPV)-infected cells cultured in the presence of cytosine-l-β-d-arabinofuranosyl-HCl (Ara C). In the immunofluorescence test, the specific antigen reacting with these MAbs was restricted almost entirely to the nucleus in CPV-infected cells in the presence of Ara C at any time. On the other hand, in CPV-infected cells in the absence of Ara C, the antigen was first detectable within the nucleus 2 h after infection and migrated to the cytoplasm as the infection proceeded. On immuno- blotting, only one component with an M r of 27K was detected 2 to 24 h after infection of cells with CPV in either the presence or absence of Ara C. The antigen was also detected in vaccinia virus-infected cells, but not in any mock-infected cells nor in any virions purified from infected cells. These results suggest that the antigen reacting with the MAbs is virus-specific and a non-structural early antigen.

The 5′ non-coding region of the genomes of 11 isolates of Murray Valley encephalitis virus from Australia and Papua New Guinea were examined by primer extension sequencing. Although the 5′ non-coding region of all isolates was found to be highly conserved, three isolates were significantly different in that they contained extra uridine residues. Two of these isolates from Papua New Guinea contained an extra uridine residue, nominally positioned after nucleotide 54, which was absent from all but one of the Australian isolates tested. This isolate (OR 156) contained a further uridine residue at the same site. These results provide further support for earlier observations on the genetic relationships between these isolates, in particular that OR 156 is more closely related to the Papua New Guinea strains than to the Australian strains.

Two monoclonal antibodies (MAbs) against p27 and one against p17 of simian immunodeficiency virus (SIV) from rhesus macaques were produced and characterized by reacting with disrupted, viral antigens on immunoblots. Human immunodeficiency virus type 1 (HIV-1), HIV-2 and SIV isolates from sooty mangabey, stump-tailed macaque, rhesus macaque and African green monkey (SIVSM ,SIVStM, SIVMAC and SIVAGM) were used for comparative analysis. The p27 monoclonal antibodies HE3 and FA2 reacted with SIVMACand SIVSM, but not with HIV-1, HIV-2,

SIVStM and SIVAGM. The p17 monoclonal antibodies reacted with SIVMACand SIVStM, but not HIV-1, HIV- 2, SIVSMand SIVAGM. The differential reactivity of these monoclonal antibodies indicated that common conserved antigenic epitopes are shared between SIVMAC and SIVSM with respect to p27 MAbs and between SIVMAC and SIVStM with respect to p17. Since these MAbs reacted differently with the SIV isolates, they are useful reagents for comparative pathogenesis studies for differentiating SIV isolates.

A novel immunohybridization assay was used to analyse the virion capsid proteins and nucleic acids of various barley yellow dwarf luteoviruses from singly and doubly infected plants. Plants singly infected with New York MAV or RPV contained only viruses indistinguishable from the parental types, but plants doubly infected with these viruses contained transcapsidated virions (MAV RNA in RPV protein capsids). The presence of transcapsidated virions was positively correlated with altered aphid transmission characteristics of MAV from the same plants. Virions with phenotypically mixed capsids (chimeric capsids containing subunits from both co-infecting viruses) were not detected in these plants using heterologous and homologous ELISAs. Transcapsidated virions were detected in mixed infections of California and New York MAV and RPV isolates and, more epidemiologically significantly, in natural doubly infected field plants. In addition, evidence for two-way transcapsida- tion was obtained using immunohybridization and aphid transmission studies from mixed infections of New York RPV and PAV isolates.

Infection of cowpea cells with cowpea mosaic virus (CPMV) is accompanied by the appearance of tubular structures containing virus-like particles which protrude from or penetrate the cell wall. Immunogold labelling of sections of infected cells using antisera against a CPMV M RNA translation products, and Protein A-gold, showed that the 58K and/or 48K tentative transport proteins of CPMV were located in or on these tubular structures. Furthermore, these proteins were detected in small electron-dense areas near the tail-end of the tubules. The possible function of these structures in virus movement from cell to cell is discussed.