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HeLa cells are used to study the life cycles of many different viruses, including the human rhinoviruses (HRV) in the family Picornaviridae. Although the natural targets of HRV are human bronchial epithelial cells (hBE), it is generally more difficult to obtain and maintain the relevant primary cell cultures, relative to HeLa cells. Given that the HRV are now identified as a major cause of human asthma exacerbations, it becomes important to document how much of the virus biology learned from HeLa cells is common also to natural primary cells. When compared directly in matched infections using A01a virus, the kinetics of RNA replication, the synthesis and processing of viral proteins and the general subcellular localization of key non-structural proteins were resembled in hBE and HeLa cells. Viral-induced shutoff of host cell processes (e.g. nucleo-cytoplasmic trafficking) was also comparable.

HeLa and 16HBE14o(-) bronchial epithelium cells infected with human rhinovirus serotype 14 (HRV14) were found to exhibit typical apoptotic morphological alterations, such as cell contraction and nuclear condensation. These events coincided with high-molecular-weight DNA fragmentation, activation of caspase-9 and caspase-3 and poly(ADP-ribose) polymerase cleavage. Caspase activation was preceded by cytochrome c translocation from the mitochondria to the cytoplasm, indicating that apoptosis caused by HRV14 infection was triggered predominantly via the mitochondrial pathway. Apoptosis did not affect HRV14 replication per se, but it facilitated the release of newly formed virus from cells. As apoptosis was fully induced at the time of maximal accumulation of progeny HRV14, it is postulated that apoptosis contributed to the destabilization of the cell and facilitated viral progeny release.

HeLa cells infected with radioactive poliovirus type 2 were disrupted with ultrasonic treatment, followed by addition of a non-ionic detergent. Two types of virus particles were found to sediment at 80 to 90% the rate of native virus. The first of these appeared to be a complex of native virus particles and membrane components, since treatment with 0-2% SDS released infectious native particles. The second was non-infectious and its sedimentation rate was not greatly altered by SDS. One hour after infection this non-infectious particle was the major product of cell-mediated eclipse. We have confirmed that 10 to 30 mg-g/ml S-7, a substituted thiopyrimidine, blocks infection of cells by poliovirus in a specific manner. Analysis of cells infected with radioactive poliovirus type 2 in the presence of S-7 showed that the virus particles remained as the complex which can be disrupted with SDS. In addition to blocking cell-mediated eclipse, S-7 stabilizes poliovirus against heat inactivation in vitro at the same concentrations which block infection. This action resembles the effect of 10-2 M-glutathione, which is also known to block cell-mediated eclipse of poliovirus.

The reduced yields of rhinovirus type 2 at temperatures above 37 degrees C were shown to result from the degradation of virus-induced RNA, leaving little RNA available for inclusion into mature infectious virions. The degradation occurred about 6 h p.i., and appeared to be selectively effecting the single-stranded species. Lysosomal nucleases do not appear to have a role in this supra-optimal degradation.

We were able to initiate a persistent infection (PI) in HeLa cells with a temperature-sensitive (ts-) mutant of rhinovirus type 2 (TS-1), but not with the corresponding wildtype (wt) virus. The ability to initiate a PI may be related to the multiplicity of infection. Persistence was established at 37 degrees C but not at 32 degrees C and the virus isolated from the PI was no longer temperature-sensitive. Infectious virus was continually produced at low levels throughout the course of the PI and cell cultures underwent multiple episodes of partial destruction (crisis) and subsequent recovery. PI virus and the initiating virus were neutralized to the same extent by hyperimmune polyclonal TS-1 antiserum indicating that no significant change had occurred with respect to serological type. The presence of either interferon or virus-related interfering activity could not be demonstrated in the PI cultures. Superinfection experiments in cells that were 'cured' of PI virus indicated the selection of a cell population during persistence that could no longer support the growth of homologous-type virus. This effect became less pronounced upon further passage of the cured cells. When compared with the wt and ts viruses, the PI virus yielded comparable amounts of infectious virus in HeLa cells but with decreased synthesis of RNA.

The polyadenylation of rhinovirus-specified RNA in aneuploid HeLa cells and diploid human embryo lung (HEL) cells has been investigated. Polyadenylation occurs at a faster rate and more extensively to virus-specified RNA in infected HEL cells compared with HeLa cells. Poly(A) is added post-transcriptively in both cell types. Furthermore the addition of adenosine or poly(A) during virus multiplication increases the polyadenylation of virus single-stranded RNAs in both cell types, but to a greater extent in HeLa cells.

The mechanism of action of a new anti-rhinovirus compound, dichloroflavan, was examined using a variety of techniques. Dichloroflavan was shown to bind to the highly sensitive rhinovirus type 1B (50% inhibitory concentration 0.007 microM) and at a much lower level to the insensitive rhinovirus type 4 (50% inhibitory concentration greater than 25 microM). Binding of the compound to rhinovirus 1B was accompanied by a reduction (about 0.5 log10 units) in virus infectivity which could be restored by treatment with chloroform. Maximum inhibition of virus yield (about 2 log10 units) occurred only when compound and virus were added together, but some inhibition (about 0.7 log10 units) occurred even when the compound was added near the end of a single cycle of replication. This late reduction in infectivity could be abolished by treating with chloroform, and was therefore caused by the compound binding to progeny virus. The compound did not interfere with adsorption of virus to cells, nor with uncoating of the viral RNA. However, little or no viral RNA and protein synthesis occurred in the presence of dichloroflavan provided that the compound was added with the virus. Addition of the compound after virus adsorption had no effect on either viral RNA or protein synthesis. These results indicate that dichloroflavan binds directly to rhinovirus 1B and appears to prevent virus replication at a point immediately after uncoating of the viral RNA.

The effect of concanavalin A (Con A) on the course of early infection of HeLa cells with purified radioactive human rhinovirus type 2 (HRV-2) or poliovirus type 2 (P-2) has been examined. Several early steps in infection were inhibited before the uncoating of parental virus. Con A, at 100 mug/ml, reduces attachment of virus when added to cells before infection. Con A also detectably slows the normal progression of adsorbed virus to tightly bound forms characterized, in the case of HRV-2, by resistance to elution by EDTA, or in a case of P-2, by isolation of a characteristic virus-membrane complex. When Con A is added together with either virus, it also inhibits cell-mediated eclipse of infectivity and the formation of non-infective subviral particles.