Effects of the adenovirus 14.7K protein, an inhibitor of TNF cytolysis, on the pathogenesis of viral infections

Abstract

The early region 3 (E3) of adenovirus (Ad) encodes a number of proteins with the potential to modulate the host immune response to infection. One of these, the Ad 14.7K protein, functions as a general inhibitor of tumor necrosis factor-{dollar}\alpha{dollar} (TNF-{dollar}\alpha{dollar})-mediated cytolysis in tissue culture assays. The studies presented in this thesis were designed to examine the effect of antagonism of TNF cytolysis by Ad 14.7K on viral pathogenicity in vivo.;We chose to study the effects of the Ad 14.7K, in isolation from other Ad immunomodulatory proteins, in a murine pneumonia model, and toward this end we cloned the l4.7K coding sequence into a vaccinia virus (VV) vector. Because it had already been demonstrated that locally produced TNF-{dollar}\alpha{dollar} has a pronounced attenuating effect on VV virulence, for some experiments we cloned the Ad2 14.7K gene into a VV vector which also contained the gene for murine TNF-{dollar}\alpha{dollar}, in order to maximize the likelihood of detecting an antagonistic relationship between the two proteins. The results of intranasal infections of BALB/c mice with these VV recombinants indicate that expression of Ad 14.7K increases the virulence of VV carrying the TNF-{dollar}\alpha{dollar} gene. This was demonstrated by increased mortality, morbidity, pulmonary pathology, and viral titers in lung tissue following, infection with VV co-expressing the 14.7K gene and TNF-{dollar}\alpha{dollar}, as compared to the control virus expressing TNF-{dollar}\alpha{dollar} alone. The biologic effects of the Ad 14.7K protein on VV pathogenesis were found to require concomitant virus-directed expression of TNF-{dollar}\alpha{dollar}, providing further evidence that the Ad 14.7K is enhancing VV pathogenicity through antagonism of TNF-{dollar}\alpha{dollar}.;Infections of C.B-17 SCID mice with the VV recombinants described above have demonstrated that mature, functional T or B lymphocytes are dispensable for both the attenuation by TNF and for its reversal by the Ad 14.7K. Expression of TNF by the recombinant VV altered the disease course and manifestations of illness, from one of early deaths due to a VV-induced pneumonia (no TNF) to one of late mortality due to a disseminated VV infection (with TNF). SCID mice infected with VV expressing both 14.7K and TNF developed, and succumbed to, a disseminated VV infection at least three weeks earlier than mice infected with VV expressing TNF alone.;Several transgenic mouse lines were constructed by Gyorgy Fejer in Dr. Horwitz's lab to contain the entire Ad2 E3 coding region under transcriptional control of its native promoter, and this thesis includes an initial characterization of the E3 expression pattern in various organs. Expression of E3 transcripts was examined by PCR amplification of reverse-transcribed total RNA, using primer pairs specific for each of the E3 mRNA's. The expression was measured basally, as well as following induction of the E3 promoter via intravenous or intraperitoneal injection of bacterial lipopolysaccharide. These studies may provide a means of upregulating transgene expression prior to infection with various pathogens, in order to examine the effects of the Ad E3 genes on pathogenesis, and may also have implications for bacterial superinfection in the context of a persistent adenovirus infection. (Abstract shortened by UMI.).