Avian sarcoma and leukosis virus cytopathic effect in the absence of TVB death domain signaling

The cytopathic effect (CPE) seen with some subgroups of avian sarcoma and leukosis virus (ASLV) is associated with viral Env activation of the death-promoting activity of TVB (a tumor necrosis factor receptor-related receptor that is most closely related to mammalian TNF-related apoptosis-inducing ligand [TRAIL] receptors) and with viral superinfection leading to unintegrated viral DNA (UVD) accumulation, which is presumed to activate a cellular DNA damage response. In this study, we employed cells that express signaling-deficient ASLV receptors to demonstrate that an ASLV CPE can be uncoupled from the death-promoting functions of the TVB receptor. However, these cell-killing events were associated with much higher levels of viral superinfection and DNA accumulation than those seen when the virus used signaling-competent TVB receptors. These findings suggest that a putative cellular DNA damage response that is activated by UVD accumulation might act in concert with the death-signaling pathways activated by Env-TVB interactions to trigger cell death. Such a model is consistent with the well-established synergy that exists between TRAIL-signaling pathways and DNA damage responses which is currently being exploited in cancer therapy regimens.     

Cytocidal effect caused by the envelope glycoprotein of a newly isolated avian hemangioma-inducing retrovirus.

We isolated a field strain of avian hemangioma retrovirus (AHV) which induces a cytopathic effect (CPE) on cultured avian and mammalian cells shortly after infection. The kinetics of cell killing were dependent on the multiplicity of infection. The CPE on avian and mammalian cells was independent of virus replication, because UV-irradiated virus led to cell death as well. Biochemical and genetic experiments indicated that AHV env gene products were responsible for the CPE. Partially purified AHV envelope glycoproteins (gp85), but not those of the Rous sarcoma virus Prague C strain, induced a CPE. Rous-associated virus type 1, in which the env region was replaced by the AHV gp85 region, induced a CPE on avian and mammalian cultured cells. Therefore, we suggest that CPE is induced by AHV via interaction between viral gp85 and the cell membrane. This mode of CPE is unique among avian sarcoma-leukemia viruses.     

Relationship of avian retrovirus DNA synthesis to integration in vitro.

An in vitro integration system derived from avian leukosis virus-infected cells supports both intra- and intermolecular integration of the viral DNA. In the absence of polyethylene glycol, intramolecular integration of viral DNA molecules into themselves (autointegration) was preferred. In the presence of polyethylene glycol, integration into an exogenously supplied DNA target was greatly promoted. Analysis of integration intermediates revealed that the strand transfer mechanisms of both reactions were identical to those of retroviruses and some transposons: each 3' end of the donor molecule is joined to a 5' end of the cleaved target DNA. The immediate integration precursor appears to be linear viral DNA with the 3' ends shortened by 2 nucleotides. Finally, in the avian system, most cytoplasmic viral DNA appears to be incomplete and further DNA synthesis is required for integration in vitro.     

Cytotoxicity of a replication-defective mutant of herpes simplex virus type 1.

Replication-defective mutants of herpes simplex virus type 1 (HSV-1) may prove useful as vectors for gene transfer, particularly to nondividing cells. Cgal delta 3 is an immediate-early gene 3 (IE 3) deletion mutant of HSV-1 that expresses the lacZ gene of Escherichia coli from the human cytomegalovirus immediate-early control region but does not express viral early or late genes. This vector was able to efficiently infect and express lacZ in cells refractory to traditional methods of gene transfer. However, 1 to 3 days postinfection, Cgal delta 3 induced cytopathic effects (CPE) in many cell types, including neurons. In human primary fibroblasts Cgal delta 3 induced chromosomal aberrations and host cell DNA fragmentation. Other HSV-1 strains that caused CPE, tested under conditions of viral replication-inhibition, included mutants of the early gene UL42, the virion host shutoff function, single mutants of IE 1, IE 2, and IE 3, and double mutants of IE 3 and 4 and IE 3 and 5. Inhibition of viral gene expression by UV irradiation of virus stocks or by preexposure of cells to interferon markedly reduced the CPE. We conclude from these studies that HSV-1 IE gene expression is sufficient for the induction of CPE, although none of the five IE gene products appear to be solely responsible. After infection of human fibroblasts with Cgal delta 3 at a low multiplicity of infection, we were able to recover up to 6% of the input virus 2 weeks later by a superinfection-rescue procedure, even though the virally transduced human cytomegalovirus-lacZ transgene was not expressed at this time. It is therefore likely that inhibition or inactivation of viral IE gene expression, either for establishing latency or for the long-term transduction of foreign genes by HSV-1 vectors, is essential to avoid the death of infected cells.     

Suppression of injuries caused by a lytic RNA virus (mengovirus) and their uncoupling from viral reproduction by mutual cell/virus disarmament

Viruses often elicit cell injury (cytopathic effect [CPE]), a major cause of viral diseases. CPE is usually considered to be a prerequisite for and/or consequence of efficient viral growth. Recently, we proposed that viral CPE may largely be due to host defensive and viral antidefensive activities. This study aimed to check the validity of this proposal by using as a model HeLa cells infected with mengovirus (MV). As we showed previously, infection of these cells with wild-type MV resulted in necrosis, whereas a mutant with incapacitated antidefensive ("security") viral leader (L) protein induced apoptosis. Here, we showed that several major morphological and biochemical signs of CPE (e.g., alterations in cellular and nuclear shape, plasma membrane, cytoskeleton, chromatin, and metabolic activity) in cells infected with L(-) mutants in the presence of an apoptosis inhibitor were strongly suppressed or delayed for long after completion of viral reproduction. These facts demonstrate that the efficient reproduction of a lytic virus may not directly require development of at least some pathological alterations normally accompanying infection. They also imply that L protein is involved in the control of many apparently unrelated functions. The results also suggest that the virus-activated program with competing necrotic and apoptotic branches is host encoded, with the choice between apoptosis and necrosis depending on a variety of intrinsic and extrinsic conditions. Implementation of this defensive suicidal program could be uncoupled from the viral reproduction. The possibility of such uncoupling has significant implications for the pathogenesis and treatment of viral diseases.     

Cell killing by avian leukosis viruses.

Infection of chicken cells with a cytopathic avian leukosis virus resulted in the detachment of killed cells from the culture dish. The detached, dead cells contained more unintegrated viral DNA than the attached cells. These results confirm the hypothesis that cell killing after infection with a cytopathic avian leukosis virus is associated with accumulation of large amounts of unintegrated viral DNA. No accumulation of large amounts of integrated viral DNA was found in cells infected with cytopathic avian leukosis viruses.     

Mutations of a residue within the polyproline-rich region of Env alter the replication rate and level of cytopathic effects in chimeric avian retroviral vectors

Previous attempts to extend the host range of the avian sarcoma/leukosis virus (ASLV)-based RCASBP vectors produced two viral vectors, RCASBP M2C (4070A) and RCASBP M2C (797-8), which replicate using the amphotropic murine leukemia virus 4070A Env protein (2). Both viruses were adapted to replicate efficiently in the avian cell line DF-1, but RCASBP M2C (4070A) caused extensive cytopathic effects (CPE) in DF-1 cells whereas RCASBP M2C (797-8) induced low levels of CPE. The two viruses differed only at amino acid 242 of the polyproline-rich region in the surface (SU) subunit of the Env protein. In RCASBP M2C (4070A), an isoleucine replaced the wild-type proline residue, whereas a threonine residue was found in RCASBP M2C (797-8). In the present study, we show that other amino acid substitutions at position 242 strongly influence the CPE and replication rate of the chimeric viruses. There was a correlation between the amount of unintegrated linear retroviral DNA present in infected DF-1 cells and the level of CPE. This suggests that there may be a role for superinfection in the CPE. The treatment of RCASBP M2C (4070A)-infected cells with dantrolene, which inhibits the release of calcium from the endoplasmic reticulum (ER), reduced the amount of CPE seen during infection with the highly cytotoxic virus. Dantrolene treatment did not appear to affect virus production, suggesting that Ca2+ release from the ER had a role in the CPE caused by these viruses.     

The forms of tRNATrp found in avian sarcoma virus and uninfected chicken cells have structural identity but functional distinctions

We have determined the complete nucleotide sequence of the avian tRNATrp which serves as primer for avian retrovirus DNA synthesis by the viral polymerase. The sequence is identical to that reported for tRNATrp present in uninfected avian cells (Harada, F., Sawyer, R. C., and Dahlberg, J. E. (1975) J. Biol. Chem. 250, 3487-3497). Although there appears to be only a single species of tRNATrp in avian cells, two functionally different forms within the population can be distinguished. We show that the tRNATrp isolated from virions can act in vitro as an efficient suppressor for UGA. The suppressor activity is roughly 3-fold greater with viral tRNATrp than with cellular tRNATrp. In addition, it has been reported (Panet, A., Haseltine, W. A., Baltimore, D., Peters, G., Harada, F., and Dahlberg, J. E. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 2535-2539) that the viral polymerase can bind 100% of viral tRNATrp, but only 30% of cellular tRNATrp. Hence, avian retroviruses seem to selectively incorporate and utilize only one of these forms. Since the nucleotide sequence and nucleoside modifications are identical between viral and cellular tRNATrp, two conformations of avian tRNATrp may exist which can influence several biological activities of the molecule.     

Moloney Murine Leukemia Virus Integrase Protein Augments Viral DNA Synthesis in Infected Cells

Mutations in the IN domain of retroviral DNA may affect multiple steps of the virus life cycle, suggesting that the IN protein may have other functions in addition to its integration function. We previously reported that the human immunodeficiency virus type 1 IN protein is required for efficient viral DNA synthesis and that this function requires specific interaction with other viral components but not enzyme (integration) activity. In this report, we characterized the structure and function of the Moloney murine leukemia virus (MLV) IN protein in viral DNA synthesis. Using an MLV vector containing green fluorescent protein as a sensitive reporter for virus infection, we found that mutations in either the catalytic triad (D184A) or the HHCC motif (H61A) reduced infectivity by approximately 1,000-fold. Mutations that deleted the entire IN (DeltaIN) or 34 C-terminal amino acid residues (Delta34) were more severely defective, with infectivity levels consistently reduced by 10,000-fold. Immunoblot analysis indicated that these mutants were similar to wild-type MLV with respect to virion production and proteolytic processing of the Gag and Pol precursor proteins. Using semiquantitative PCR to analyze viral cDNA synthesis in infected cells, we found the Delta34 and DeltaIN mutants to be markedly impaired while the D184A and H61A mutants synthesized cDNA at levels similar to the wild type. The DNA synthesis defect was rescued by complementing the Delta34 and DeltaIN mutants in trans with either wild-type IN or the D184A mutant IN, provided as a Gag-IN fusion protein. However, the DNA synthesis defect of DeltaIN mutant virions could not be complemented with the Delta34 IN mutant. Taken together, these analyses strongly suggested that the MLV IN protein itself is required for efficient viral DNA synthesis and that this function may be conserved among other retroviruses.     

Correlation between cell killing and massive second-round superinfection by members of some subgroups of avian leukosis virus.

Avian leukosis viruses of subgroups B, D, and F are cytopathic for chicken cells, whereas viruses of subgroups A, C, and E are not. The amounts of unintegrated linear viral DNA in cells at different times after infection with cytopathic or noncytopathic viruses were determined by hybridization and transfection assays. Shortly after infection, there is a transient accumulation of unintegrated linear viral DNA in cells infected with cytopathic avian leukosis viruses. By 10 days after infection, the majority of this unintegrated viral DNA is not present in the infected cells. The transient cytopathic effect seen in these infected cells also disappears by this time. Low amounts of unintegrated linear viral DNA persist in these cells. Cells infected with noncytopathic viruses do not show this transient accumulation of unintegrated viral DNA. Cells infected with cytopathic viruses and subsequently grown in the presence of neutralizing antibody do not show the transient accumulation of unintegrated viral DNA or cytopathic effects. These results demonstrate a correlation between envelope subgroup, transient accumulation of unintegrated linear viral DNA, and transient cell killing by avian leukosis viruses. The cell killing appears to be the result of massive second-round superinfection by the cytopathic avian leukosis viruses.     

Establishment of infection by spleen necrosis virus: inhibition in stationary cells and the role of secondary infection

The relationship of two early events in the establishment of infection by avian retroviruses, the inhibition of viral DNA synthesis in stationary avian cells and the secondary infection which occurs after infection of replicating cells, was investigated. When neutralizing antibody to spleen necrosis virus was used to prevent secondary infection, the amount of unintegrated linear spleen necrosis virus DNA detected was much lower in infected stationary cells than in infected replicating cells. The amount of unintegrated linear spleen necrosis virus DNA in stationary cells was less than one copy per cell even at high multiplicities of infection. Viral DNA synthesis resumed after stimulation of the cells to replicate. The time of this viral DNA synthesis was closely correlated with renewed cellular DNA synthesis. In addition, blocking secondary infection of replicating cells prevented the rate of virus production from reaching the high levels usually associated with a normal productive infection by SNV. Virus production increased if secondary infection was allowed. However, this rise in virus production was not proportional to the amounts of viral DNA integrated after secondary infection.     

Evidence for two points of restriction in the expression of adenovirus type 2 in cultured epidermal keratinocytes.

Cultures of epidermal keratinocytes contain two populations of cells, a basal undifferentiated population and a suprabasal terminally differentiated population. When exposed to wild-type adenovirus type 2 (wtAd2), the suprabasal cells are positive by immunofluorescence for capsid antigen and exhibit cytopathic effects (CPE) (R.F. LaPorta, and L.B. Taichman, Virology 110:137-146, 1981). The basal cells, although infected, are not positive for capsid antigen and do not display CPE. Despite CPE and capsid antigens in suprabasal cells, yields of virus from the entire culture are very low (10 PFU per cell). These observations suggest that Ad2 expression is restricted at different times in the viral life cycle in basal and suprabasal cells. To test this hypothesis, we isolated host range (hr) mutants of Ad2 on two lines of squamous cell carcinoma (SCC) keratinocytes which were shown to be restrictive for wtAd2 replication. The hrAd2 mutants produced high yields of progeny virus in epidermal cell cultures (500 to 600 PFU per cell). However, the pattern of CPE induction in these cultures was like that produced by wtAd2, i.e., basal cells were CPE negative and suprabasal cells were CPE positive. The high yield of hrAd2 progeny indicated that the restriction present in suprabasal cells was overcome. However, the failure of hrAd2 mutants to induce CPE in basal cells indicated that the hrAd2 mutants remain restricted in the basal population and supported our hypothesis that a second and distinct restriction exists in basal keratinocytes.     

Mutational analysis of the hypervariable region of hepatitis e virus reveals its involvement in the efficiency of viral RNA replication

The RNA genome of the hepatitis E virus (HEV) contains a hypervariable region (HVR) in ORF1 that tolerates small deletions with respect to infectivity. To further investigate the role of the HVR in HEV replication, we constructed a panel of mutants with overlapping deletions in the N-terminal, central, and C-terminal regions of the HVR by using a genotype 1 human HEV luciferase replicon and analyzed the effects of deletions on viral RNA replication in Huh7 cells. We found that the replication levels of the HVR deletion mutants were markedly reduced in Huh7 cells, suggesting a role of the HVR in viral replication efficiency. To further verify the results, we constructed HVR deletion mutants by using a genetically divergent, nonmammalian avian HEV, and similar effects on viral replication efficiency were observed when the avian HEV mutants were tested in LMH cells. Furthermore, the impact of complete HVR deletion on virus infectivity was tested in chickens, using an avian HEV mutant with a complete HVR deletion. Although the deletion mutant was still replication competent in LMH cells, the complete HVR deletion resulted in a loss of avian HEV infectivity in chickens. Since the HVR exhibits extensive variations in sequence and length among different HEV genotypes, we further examined the interchangeability of HVRs and demonstrated that HVR sequences are functionally exchangeable between HEV genotypes with regard to viral replication and infectivity in vitro, although genotype-specific HVR differences in replication efficiency were observed. The results showed that although the HVR tolerates small deletions with regard to infectivity, it may interact with viral and host factors to modulate the efficiency of HEV replication.     

Modifications in the binding domain of avian retrovirus envelope protein to redirect the host range of retroviral vectors.

On the basis of theoretical structural and comparative studies of various avian leukosis virus SU (surface) envelope proteins, we have identified four small regions (I, II, III, and IV) in their receptor-binding domains that could potentially be involved in binding to receptors. From the envelope gene of an avian leukosis virus of subgroup A, we have constructed a set of SU mutants in which these regions were replaced by the coding sequence of FLA16, a 16-amino-acid RGD-containing peptide known to be the target for several cellular integrin receptors. Helper-free retroviral particles carrying a neo-lacZ retroviral vector were produced with the mutant envelopes. SU mutants in which regions III and IV were substituted yielded normal levels of envelope precursors but were not detectably processed or incorporated in viral particles. In contrast, substitutions in regions I and II did not affect the processing and the viral incorporation of SU mutants. When FLA16 was inserted in region II, it could be detected with antibodies against FLA16 synthetic peptide, but only when viral particles were deglycosylated. Viral particles with envelopes mutated in region I or II were able to infect avian cells through the subgroup A receptor at levels similar to those of the wild type. When viruses with envelopes containing FLA16 peptide in region II were applied to plastic dishes, they were found to promote binding of mammalian cells resistant to infection by subgroup A avian leukosis viruses but expressing the integrins recognized by FLA16. Deglycosylated helper-free viruses obtained by mild treatment with N-glycosidase F have been used to infect these mammalian cells, and infections have been monitored by neomycin selection. No neomycin-resistant clones could be obtained after infection by viruses with wild-type envelopes. Conversely, colonies were obtained after infection by viruses with envelopes bearing FLA16 in region II, and the genome of the retroviral vector was found correctly integrated in cell DNA of these colonies. By using a blocking peptide containing the minimal adhesive RGD sequence contained in FLA16, we have shown that preincubation of target cells could specifically inhibit infection by viruses with FLA16.     

Replication of herpesvirus DNA. V. Maturation of concatemeric DNA of pseudorabies virus to genome length is related to capsid formation.

The maturation of pseudorabies virus DNA from the replicative concatemeric form to molecules of genome length was examined using nine DNA+ temperature-sensitive mutants of pseudorabies virus, each belonging to a different complementation group. At the nonpermissive temperature, cells infected with each of the mutants synthesized concatemeric DNA. Cleavage of the concatemeric DNA to genome-length viral DNA was defective in all the DNA+ ts mutants tested, indicating that several viral gene products are involved in the DNA maturation process. In none of the ts mutant-infected cells were capsids with electron-dense cores (containing DNA) formed. Empty capsids with electron-translucent cores were, however, formed in cells infected with six of the nine temperature-sensitive mutants; in cells infected with three of the mutants, no capsid assembly occurred. Because these three mutants are deficient both in maturation of DNA and in the assembly of viral capsids, we conclude that maturation of viral DNA is dependent upon the assembly of capsids. In cells infected with two of the mutants (tsN and tsIE13), normal maturation of viral DNA occurred after shiftdown of the cells to the permissive temperature in the presence of cycloheximide, indicating that the temperature-sensitive proteins involved in DNA maturation became functional after shiftdown. Furthermore, because cycloheximide reduces maturation of DNA in wild-type-infected cells but not in cells infected with these two mutants, we conclude that a protein(s) necessary for the maturation of concatemeric DNA, which is present in limiting amounts during the normal course of infection, accumulated in the mutant-infected cells at the nonpermissive temperature. Concomitant with cleavage of concatemeric DNA, full capsids with electron-dense cores appeared after shiftdown of tsN-infected cells to the permissive temperature, indicating that there may be a correlation between maturation of DNA and formation of full capsids. The number of empty and full capsids (containing electron-dense cores) present in tsN-infected cells incubated at the nonpermissive temperature, as well as after shiftdown to the permissive temperature in the presence of cycloheximide, was determined by electron microscopy and by sedimentation analysis in sucrose gradients. After shiftdown to the permissive temperature in the presence of cycloheximide, the number of empty capsids present in tsN-infected cells decreased with a concomitant accumulation of full capsids, indicating that empty capsids are precursors to full capsids.