Infective centers

Targeted tuberculin testing and treatment of latent infection. Centers For Disease Control and Prevention MMWR 2000 49 1-54. 

A encodes six regulatory protein cl, ell, cIII, cro, N, and Q (table 30.2). The developmental processes accompanying infection center on the roles of these six regulatory proteins and the factors that govern the level of their expression. 

Fig. 2 a. Transfection curves for 029 DNA (closed circles) and SP02 DNA (open circles). The number of infective centers/ml is plotted against the concentration of DNA in the transfection mixture, expressed as phage equivalents/ml. Conversion factors 1 (Jtg 029 DNA = 5.0X10 phage equiv. 1 [xg SPO 2 DNA = 2.3 XIO phage equiv. (Spatz and Trautner, 1971). b. Transfection curves for SPPi DNA (Closed circles) and SP 50 DNA (open circles). The data are plotted as in Fig. 2a. Conversion factors 1 jxg SPPi DNA=2.4X10 phage equiv. 1 xg SP 50 DNA=6.0X10 phage equiv.

DNA Molecular weight (10 daltons) Exponent of Concentration Dependence Maximal Efficiency of Transfection (infective centers/phage equivalent of DNA)... 

The efficiency is expressed as the ratio of wild-type infective centers to the number of transfecting DNA molecules (phage equivalents). 

The concentration of DNA used for this comparison was adjusted to give the same number of infective centers for all three sets of experiments. 

D. Relationship between RNA Concentration and Yield of Infective Centers 109... 

In most of the test systems studied so far, the yield of infective centers obtained with isolated RNA is somewhat less than 1% of the infectivity of the virus preparation from which the RNA was isolated (Dubes, 1971). The relatively low infectivity of naked nucleic acid is attributed to the sensitivity of nucleic acids to degrading enzymes and the low efficiency of adsorption and penetration into cells of the naked RNA. 

The penetration of infectious RF-RNA, can also be measured by following the effect of RNase on infective center formation (Fig. 8). In an isotonic environment, most of the RF-RNA infectivity is lost rapidly when exposed to RNcise (Bishop and Koch, 1967 Mittelstaedt and Koch, 1974). Only those cells which have already engulfed one complete RNA molecule before RNase is added will register as an infectious center in the plaque assay. Both... 

Poly cat ion concentration Infective centers/pg RNA and time of addition --------------------------... 

After exposing cells to appropriate concentrations of polycations, all RNA is adsorbed (Fig.2). Judged by the effect of RNase on infective center formation (Fig. 8), 60 to 80% of viral RNA penetrates the cells within 15 to 20 minutes at 37° C. All RF-RNA penetrates the cells within 5 to 10 minutes... 

Fig. 9. Relationship between polycation to cell ratio and yield of infective centers. A standard dilution of viral RNA was assayed with a series of cell and polycation concentrations so as to obtain the range of polycation to cell ratios indicated and a significant number of PFU...

The mechanism whereby polycations sensitize cells for infection by viral RNA and RF-RNA has been studied in detail. A wide variety of polycations can be used to augment the infectivity of viral RNA (Table 5). The dose-response curves obtained with four different polycations show remarkable similarities (Fig. 7). As the polycation concentrations increase, there is a 100,000-fold rise in the yield of infective centers, which, however, falls sharply when the polycation concentration is increased over a certain limit. 

We discussed above the effect of polycations on RNA adsorption. A comparison of the data of Figs. 2 and 8 shows that a 3 to 10-fold concentration of DEAE-dextran is required to obtain a maximal yield of infective centers compared to a maximal degree of RNA adsorption. These results indicate that polycations have a dual effect on the interaction of RNA with cells exposure of suspended cells to low polycation concentrations results in optimal adsorption of RNA, but high polycation concentrations are required for an optimal RNA infectivity. These observations pose a number of questions ... 

Fig. 11. Relationship between RNA concentration and yield of infective centers as determined with the agar cell suspension plaque assay. A viral RNA preparation was diluted and various amounts of RNA incubated with 2X10 cells pre-exposed to optimal concentrations of polycations. At the higher RNA concentrations, infective centers induced by RNA had to be diluted prior to plating so that individual plaques could be counted. The ordinate indicates the yield of infective centers...

HLA virus (5 [xg/ml) and their sensitivity to infection by isolated RNA was analyzed. We found (see Table 5) the cells exposed to HLA virus for one minute at 37° C prior to addition of single- or double-stranded poliovirus RNA yielded 100 times more infective centers after 15 min incubation at 37° C than untreated cells (Breindl and Koch, 1972). This result reveals that the poliovirus coat proteins are able to sensitize HeLa cells to infection by viral RNA. 

---