- Volume 51, Issue 2, 1980

Washed, non-viable sperm from AKR and NZB mice contain infectious murine type C viruses (MuLV), whereas similarly treated sperm from Balb/c and probably C57Bl/6 mice do not. Interaction of washed, viable AKR, Balb/c and NZB sperm with tissue culture cells leads to a transfer of infectious MuLV to these cells. The most likely mechanism is infection by virus closely associated with the sperm. Another possible mechanism is the introduction of proviral DNA. These experiments indicate that type C viruses can be found with mouse sperm. They suggest sperm could horizontally transmit these MuLV to animals during copulation and to the germ line of mice during penetration of ova.

Two virus-induced polypeptides are preferentially released into the culture medium of owl monkey kidney cells productively infected with herpesvirus saimiri (HVS). These polypeptides have apparent mol. wt. of 160 × 103 and 28 × 103 and are the major virus-induced polypeptides in a membrane fraction from infected cells. Both these excreted polypeptides co-migrate with virus structural proteins and are selectively released from enveloped virus particles by treatment with Nonidet-P40.

In hybridization experiments, using complementary DNA (cDNA) copies of the two genome parts of tomato black ring virus (TBRV RNA-1 and RNA-2), no sequence homology between the two RNA species was detected.

When tobacco mesophyll protoplasts were inoculated with purified middle component particles, which contain only RNA-2, no replication of TBRV RNA could be detected. However, when they were inoculated with purified bottom component particles, which contain only RNA-1, extracts made 24 or 48 h later contained RNA that had the same mobility as RNA-1 in polyacrylamide-agarose gels, and that hybridized to cDNA copies of RNA-1. Thus RNA-1 can replicate in protoplasts that do not contain RNA-2. Moreover, this RNA-1 was capable, when mixed with nucleoprotein particles containing RNA-2, of inducing the formation of local lesions in Chenopodium amaranticolor leaves, and therefore was intact and attached to the genome-linked protein. The genome-linked protein of nepoviruses is probably virus-coded, and its production in protoplasts inoculated with bottom component particles therefore suggests that RNA-1 contains the gene for this protein.

A study was made of the proteins synthesized in cowpea protoplasts infected with cowpea mosaic virus (CPMV). Protoplasts were labelled with 35S-methionine at different times during the infection cycle and the proteins synthesized were analysed by SDS-polyacrylamide slab gel electrophoresis of different subcellular fractions. Since no shut-off of host protein synthesis occurred, virus-specific protein synthesis had to be distinguished from a high background of protoplast proteins. Eleven polypeptides with mol. wt. of 170, 130, 112, 110, 87, 84, 68, 37, 30, 24 and 23 (all × 103) were detected whose synthesis was either induced or stimulated by CPMV infection. No differences were observed between the electrophoretic patterns of samples from uninfected and CPMV-infected protoplasts during the first 8 h of infection (latent phase). The 170 × 103 and 30 × 103 species were the first virus-related polypeptides detectable by labelling between 9 and 15 h after inoculation. From about 16 h after inoculation, all other virus-related proteins appeared to be synthesized at increasing rates. The polypeptides with mol. wt. of 170, 130, 112, 110, 68, 37 and 23 (all × 103) could be designated as virus-induced; the others may well be host-coded proteins, the synthesis of which is stimulated by CPMV infection. Although CPMV replication is known to occur in close association with virus-induced cytopathic membrane structures the 112 × 103 and 68 × 103 mol. wt. polypeptides were the only virus-related proteins specifically found in the particulate fractions of the protoplasts. From the 11 virus-related proteins, the 37 × 103, 24 × 103 and 23 × 103 mol. wt. proteins were identified as virus coat proteins. The 170 × 103 and 30 × 103 mol. wt. polypeptides also appeared to be virus-specific and to be coded for by CPMV bottom component RNA.

Inoculation of cowpea protoplasts with purified middle component of cowpea mosaic virus (CPMV) did not result in the production of newly induced virus-related proteins. In contrast, infection of protoplasts with purified bottom component produced virus-related polypeptides with mol. wt. 170×103, 112 × 103, 110 × 103, 84 × 103 and 30 × 103, but not the virus capsid proteins. These results suggest that CPMV B-RNA specifies one or more early functions, one of which is the replicase function, while m-RNA encodes the virus coat proteins. Attempts to elucidate the mechanism by which the virus-specific proteins are synthesized were inconclusive. No precursor-product relations could be demonstrated and virus subgenomic messengers were not observed. Some kind of processing was nonetheless found to occur with the small virus capsid protein:several discrete oligopeptides were successively cleaved from this protein after assembly into the virion particle.

The immune electron microscopic technique which involves pre-coating electron microscope grids with protein A before coating them with the specific antiserum, has been found suitable for detecting isometric insect and plant viruses. With the three virus-antibody combinations tested, the optimum antiserum dilution for protein A plus antiserum treatment was found to be 1:100 or less, whereas in the case of grids treated with antiserum alone it ranged from 1:1000 to 1:2000 although the titre of the antisera ranged from 1:512 to 1:4096. The increase in the number of particles on grids treated with protein A plus antiserum over those treated with antiserum alone, at each optimal antiserum dilution, was 25.7-fold (sugarcane mosaic virus), 2.1-fold (tobacco mosaic virus) and 1.6- and 2.4-fold (Erysimum latent virus — two different sap dilutions). Protein A plus antiserum-coated grids can be stored for up to 6 months at 4 °C, but not at room temperature or in a desiccator, and still retain about 25% of their activity, sufficient to detect any virus using the electron microscope. Antisera preserved in glycerol can be used successfully for detecting viruses by immune electron microscopy.