- Volume 67, Issue 9, 1986

The presence of human papillomavirus (HPV) type 16 DNA in biopsies from precancerous lesions and from early lesions of human cervical cancer, and the integration of virus DNA into host cell DNA were analysed by dot blot and Southern blot hybridizations. HPV 16 DNA was detected in 23% of mild dysplasias, 32% of moderate dysplasias, 55% of severe dysplasias and 62% of carcinomas in situ by dot blot hybridization. Digestion of the DNA with restriction enzymes PstI and BamHI followed by Southern blot analysis revealed the presence of some typical restriction fragments of HPV 16 DNA in most virus-positive samples. In addition, we detected submolar fragments which might represent virus-cell junction sequences in 86% of dysplasias, suggesting that the integration of HPV 16 DNA could occur in the precancerous stage.

This study was undertaken to determine the effect of micromolar concentrations of delta-9-tetrahydrocannabinol (Delta-9-THC) on herpes simplex virus type 2 (HSV-2) replication in vitro. Virus-infected Vero cells pretreated for 24 h with 10−5 m- or 10−6 m-Delta-9-THC yielded 100-fold increases in infectious extracellular virus. Transmission electron microscopy of drug-treated cells revealed plasma membrane dissolution, distension of the smooth and rough endoplasmic reticulum, and the appearance of macrovacuoles in the cytoplasm containing aggregates of virus. These results suggest that Delta-9-THC enhances the release of HSV-2 by perturbing cellular membranes in virus-infected cells.

Herpes simplex virus type 1 and a fluorescein-labelled lectin (wheat germ agglutinin) were selectively transported to nerve cell bodies located in the inner compartment of a two-chamber tissue culture system after the application of virus or lectin to the neuritic processes in the outer culture compartment. Taxol, which stabilizes and alters intracellular arrangements of microtubules, and nocodazole, which disrupts microtubules, both inhibited this retrograde axonal transport of viral particles and lectin. The transport was also inhibited by erythro-9-3-(2-hydroxynonyl)adenine (EHNA), which blocks ATPases. However, EHNA was also an effective inhibitor of infection with the virus in non-neuronal cells (GMK AH-1). The nature of the action(s) of EHNA on neuritic transport of the virus is therefore less clear.

Reduced temperature has been shown to block the cell surface expression of Sendai virus haemagglutinin-neuraminidase (HN) and fusion (F0) glycoproteins at different steps of their intracellular transport. At 20 °C, HN was confined to the rough endoplasmic reticulum or cis Golgi compartment, while F0 acquired complete resistance to digestion by endo-β-N-acetylglucosaminidase-H and therefore was blocked at a more distal location in the pathway of cell surface expression. The significance of these results for different pathways of transport to the cell surface is discussed.

In vivo immunization of BALB/c mice with poliovirus type 1, strain Mahoney, or with its purified polypeptides resulted in 0.2 to 0.5 antigen-specific hybridoma microcultures per 106 spleen cells. Stimulation of spleen cells from mice immunized with poliovirus or with its polypeptides in vitro with poliovirus 6 days prior to fusion with the myeloma cells led to a six- to 20-fold increase in the number of positive microcultures, i.e. after stimulation the yield of poliovirus-specific hybridomas was up to 3.8 antigen-specific microcultures per 106 spleen cells. The in vitro stimulation of spleen cells primed in vivo was demonstrated by the detection of poliovirus-specific antibody-producing cells 6 days after in vitro cultivation in the presence of poliovirus as antigen. Only spleen cells stimulated under these conditions in vitro gave rise to specific antibody-producing cells and yielded antigen-specific hybridomas after somatic hybridization.

Glycoproteins synthesized in both human (HeLa and HEp-2) and simian (Vero and BS-C-1) cell lines following infection with two different strains of respiratory syncytial virus (A2 and Long) were analysed by SDS–PAGE following immunoprecipitation with monoclonal antibodies. Minor virus strain-dependent differences in the large glycoprotein, G, and the fusion protein polypeptides F1 and F2 were observed together with minor cell line-dependent differences in the size of the F2 polypeptide. Major quantities of two glycoproteins, termed Ga (50K) and Gb (45K), were detected in A2 strain-, and to a lesser extent in Long strain-, infected simian cells. These proteins were also present in infected human cells, but in much reduced amounts. Immunoprecipitation with anti-G monoclonal antibodies demonstrated that Ga and Gb shared different epitopes with G.

A cyclic nucleotide-independent protein kinase (PK) activity has been found to be associated with purified particles of cauliflower mosaic virus. The main acceptors of phosphorylation were proteins with mol. wt. of 42000 (the capsid protein), 58000 (which may be the capsid protein precursor) and 110000 (of unknown function). Acid hydrolysis and phosphoamino acid analysis of nucleocapsid proteins phosphorylated in vitro showed that the PK catalyses the transfer of phosphate to both serine and threonine residues. Activation of the PK made the DNA more accessible to DNase I, suggesting that a modification of the structure of the capsid had occurred.

Tobacco mesophyll protoplasts were transfected with tobacco mosaic virus (TMV) RNA in an electric field using a newly devised chamber constructed with gold-coated glass panel electrodes. Up to 95% of protoplasts subjected to several DC pulses (50 µs, 550 to 800 V/cm) while suspended in 0.5 m-mannitol containing 10 µg/ml TMV RNA became infected. The treatment did not affect the viability of the protoplasts or their stability during 40 h of incubation.

Northern blot hybridization experiments with cDNAs to the four RNA species of a Yugoslavian isolate of beet necrotic yellow vein virus (BNYVV) revealed identical virus RNA patterns in root extracts from field-grown sugarbeets and sugarbeet seedlings grown in soil from rhizomania-affected fields in various regions in Germany and abroad. In contrast, in leaf extracts from mechanically infected Chenopodium quinoa, Tetragonia expansa and sugarbeet and from a naturally infected sugarbeet we observed great variations in the number, size and relative concentration of the small BNYVV RNAs, which suggests that they may undergo deletion mutations when the virus is propagated in leaf tissues.

Three of the four dsRNA components of purified beet cryptic virus (BCV) were copied into cDNA and cloned into pUC9. Clones corresponding to RNAs 1, 3 and 4 did not hybridize to each other or to RNA 2, suggesting that there is no significant sequence homology between the four dsRNA components. RNA extracted from 15 BCV-infected beet plants was analysed by Northern blotting using the cDNA clones as probes. Nine plants were found to contain RNAs 1, 3 and 4 whereas in six plants only RNAs 3 and 4 were detectable. The results are compatible with the occurrence of two different viruses. The sensitivity and specificity of the cDNA hybridization assay was greater than that of immunosorbent electron microscopy in the detection of BCVs.