Gary Ketner

Adenovirus E4 34k and E1b 55k Oncoproteins Target Host DNA Ligase IV for Proteasomal Degradation

ABSTRACT Cells infected by adenovirus E4 mutants accumulate end-to-end concatemers of the viral genome that are assembled from unit-length viral DNAs by nonhomologous end joining (NHEJ). Genome concatenation can be prevented by expression either of E4 11k (product of E4orf3) or of the complex of E4 34k (product of E4orf6) and E1b 55k. Both E4 11k and the E4 34k/E1b 55k complex prevent concatenation at least in part by inactivation of the host protein Mre11: E4 11k sequesters Mre11 in aggresomes, while the E4 34k/E1b 55k complex participates in a virus-specific E3 ubiquitin ligase that mediates ubiquitination and proteasomal degradation. The E4 34k/E1b 55k complex, but not E4 11k, also inhibits NHEJ activity on internal breaks in the viral genome and on V(D)J recombination substrate plasmids, suggesting that it may interfere with NHEJ independently of its effect on Mre11. We show here that DNA ligase IV, which performs the joining step of NHEJ, is degraded as a consequence of adenovirus infection. Degradation is dependent upon E4 34k and E1b 55k, functional proteasomes, and the activity of cellular cullin 5, a component of the adenoviral ubiquitin ligase. DNA ligase IV also interacts physically with E1b 55k. The data demonstrate that DNA ligase IV, like Mre11, is a substrate for the adenovirus-specific E3 ubiquitin ligase; identify an additional viral approach to prevention of genome concatenation; and provide a mechanism for the general inhibition of NHEJ by adenoviruses.

Interaction of adenoviral E4 and E1b products in late gene expression

The adenovirus 294R protein of E4 ORF 6 forms a physical and functional complex with the 496R protein product of E1b. The E4 294R ORF 6 protein also functions in parallel with the E4 116R ORF 3 protein in viral late protein synthesis. We have examined the roles of these three proteins and the protein complex in viral late protein synthesis and late message metabolism, by comparing the phenotypes of E4 294R-, E4 116R-, and E1b 496R- mutants to those of a series of double mutants. Our data indicate that the 294R and 116R proteins act in parallel to permit the accumulation of normal levels of unprocessed late viral RNA in the nucleus of infected cells. Both 294R and 496R function in parallel with the 116R protein in viral nuclear RNA accumulation, but do so to different degrees, suggesting that 294R and 496R may have roles apart from the functional complex in mediating accumulation of viral messages in the nucleus, or that they have nonequivalent roles within the complex. Our results are also consistent with a role for the 496R/294R protein complex in mediating efficient transport of late messages from the nucleus to the cytoplasm and/or in maintaining the stability of those messages on reaching the cytoplasm, as suggested previously (S. Pilder, M. Moore, J. Logan, and T. Shenk, 1986, Mol. Cell. Biol. 6, 470-476). Finally the 116R protein seems to act in parallel with the complex to permit normal viral DNA replication.

Both BC-Box Motifs of Adenovirus Protein E4orf6 Are Required To Efficiently Assemble an E3 Ligase Complex That Degrades p53

ABSTRACT Small DNA tumor viruses typically encode proteins that either inactivate or degrade p53. Human adenoviruses encode products, including E4orf6 and E1B55K, that do both. Each independently binds to p53 and inhibits its ability to activate gene expression; however, in combination they induce p53 degradation by the ubiquitin pathway. We have shown previously that p53 degradation relies on interactions of E4orf6 with the cellular proteins Cul5, Rbx1, and elongins B and C to form an E3 ligase similar to the SCF and VBC complexes. Here we show that, like other elongin BC-interacting proteins, including elongin A, von Hippel-Lindau protein, and Muf1, the interaction of E4orf6 is mediated by the BC-box motif; however, E4orf6 uniquely utilizes two BC-box motifs for degradation of p53 and another target, Mre11. In addition, our data suggest that the interaction of E1B55K with E4orf6 depends on the ability of E4orf6 to form the E3 ligase complex and that such complex formation may be required for all E4orf6-E1B55K functions.

Adenovirus L4-100K Assembly Protein Is a Granzyme B Substrate that Potently Inhibits Granzyme B-Mediated Cell Death

Cytotoxic lymphocytes kill virus-infected target cells and play a critical role in host recovery from viral infections. Granzyme B (GrB) is a cytotoxic lymphocyte granule protease that plays a critical role in mediating cytotoxicity. In these studies, we demonstrate that the adenovirus assembly protein L4--100K (100K) is a GrB substrate that prevents cytotoxic lymphocyte granule-induced apoptosis in infected target cells by potently inhibiting GrB. This inhibition is absolutely dependent on Asp-48 in 100K, found within a classic GrB consensus motif. 100K is the first viral protein described that exclusively targets the GrB pathway. It represents a novel class of viral protease inhibitor, in which an essential, multifunctional viral protein, which is vulnerable to specific proteolysis by GrB, expresses inhibitory function against that protease.

Vaccination with HPV16 L2E6E7 fusion protein in GPI-0100 adjuvant elicits protective humoral and cell-mediated immunity.

A vaccine comprising human papillomavirus type 16 (HPV16) L2, E6 and E7 in a single tandem fusion protein (termed TA-CIN) has the potential advantages of both broad cross-protection against HPV transmission through induction of L2 antibodies able to cross neutralize different HPV types and of therapy by stimulating T cell responses targeting HPV16 early proteins. However, patients vaccinated with TA-CIN alone develop weak HPV neutralizing antibody and E6/E7-specific T cell responses. Here we test TA-CIN formulated along with the adjuvant GPI-0100, a semi-synthetic quillaja saponin analog that was developed to promote both humoral and cellular immune responses. Subcutaneous administration to mice of TA-CIN (20 microg) with 50microg GPI-0100, three times at biweekly intervals, elicited high titer HPV16 neutralizing serum antibody, robust neutralizing titers for other HPV16-related types, including HPV31 and HPV58, and neutralized to a lesser extent other genital mucosatropic papillomaviruses like HPV18, HPV45, HPV6 and HPV11. Notably, vaccination with TA-CIN in GPI-0100 protected mice from cutaneous HPV16 challenge as effectively as HPV16 L1 VLP without adjuvant. Formulation of TA-CIN with GPI-0100 enhanced the production of E7-specific, interferon gamma producing CD8(+) T cell precursors by 20-fold. Vaccination with TA-CIN in GPI-0100 also completely prevented tumor growth after challenge with 5x10(4) HPV16-transformed TC-1 tumor cells, whereas vaccination with TA-CIN alone delayed tumor growth. Furthermore, three monthly vaccinations with 125 microg of TA-CIN and 1000 microg GPI-0100 were well tolerated by pigtail macaques and induced both HPV16 E6/E7-specific T cell responses and serum antibodies that neutralized all HPV types tested.

Deletion mutants of adenovirus 2: Isolation and initial characterization of virus carrying mutations near the right end of the viral genome

Abstract A series of eight deletion mutants of human adenovirus 2 have been isolated by a procedure which employs temperature-sensitive adenovirus mutants as helper viruses. Seven of these deletions are located in the right end of the viral genome and cover portions of early regions 3 and/or 4. The mutant with the largest deletion (807) has been separated from its helper by CsCl density gradient centrifugation. Measurements of the synthesis of viral DNA and late viral protein in 807-infected cells show that the mutant synthesizes DNA normally, and. that it produces reduced amounts of some, but not all, late proteins. These observations suggest that while the region 82.5–95 on the adenoviral genome contains no gene essential for DNA replication, it does include a gene or site whose function affects the synthesis of late proteins encoded by widely separated regions of the viral genome.

Adenovirus E4orf6 assembles with Cullin5-ElonginB-ElonginC E3 ubiquitin ligase through an HIV/SIV Vif-like BC-box to regulate p53

The adenovirus protein E4orf6 targets p53 for polyubiquitination and proteasomal degradation and is known to form a complex with the Cul5‐ElonginB‐ElonginC E3 ubiquitin ligase. However, whether Cul5 is directly responsible for the E4orf6‐mediated degradation of p53 remains unclear. By using a dominant‐negative mutant of Cul5 and silencing Cul5 expression through RNA interference, we have now demonstrated that E4orf6‐mediated p53 degradation requires Cul5. Furthermore, we have identified a lenti‐viral Vif‐like BC‐box motif in E4orf6 that is highly conserved among adenoviruses from multiple species. More importantly, we have shown that this Vif‐like BC‐box is essential for the recruitment of Cul5‐Elong‐inB‐ElonginC E3 ubiquitin ligase by E4orf6 and is also required for E4orf6‐mediated p53 degradation. E4orf6 selectively recruited Cul5 despite the lack of either a Cul5‐box, which is used by cellular substrate receptors to recruit Cul5, or a newly identified HCCH zinc‐binding motif, which is used by primate lentiviral Vif to recruit Cul5. Therefore, adenovirus E4orf6 molecules represent a novel family of viral BC‐box proteins the cellular ancestor of which is as yet unknown.—Luo K., Ehrlich, E., Xiao, Z., Zhang, W., Ketner, G., Yu X.‐F. Adenovirus E4orf6 assembles with Cullin5‐ElonginB‐ElonginC E3 ubiquitin ligase through an HIV/SIV Vif‐like BC‐box to regulate p53. FASEB J. 21, 1742–1750 (2007)

Properties of an adenovirus type 2 mutant, Ad2d/807, having a deletion near the right-hand genome terminus: Failure to help AAV replication

We have analyzed the ability of an adenovirus type 2 mutant, Ad2dl807, to support replication of adeno-associated virus (AAV). This mutant has a deletion extending from early region 3 through the late fiber gene and into early region 4. Since AAV growth does not require Ad early region 3 or the fiber gene, the Ad2dl807 mutant allows analysis of the function of Ad early region 4 in AAV growth. As determined by assay of AAV DNA, RNA, and protein synthesis as well as the number of cells producing AAV and the yield of infectious AAV particles, growth of AAV with the dl807 mutant is decreased 10- to 20-fold. The residual AAV growth is probably due to the presence of a low level of contaminating viable Ad virions in the dl807 preparations. These observations indicate that an Ad early region 4 function is required at a very early stage of AAV DNA replication to allow amplification of duplex replicating (RF) DNA. Thus growth of AAV offers an independent probe for adenovirus early region 4 functions.

Vectored antibody gene delivery protects against Plasmodium falciparum sporozoite challenge in mice

Significance Malaria caused by Plasmodium falciparum results in the death of between 500,000 and 800,000 children per year and thus presents a major infectious disease threat to public health. Antibodies directed against the circumsporozoite protein of the infectious form of the parasite can prevent P. falciparum infection. Nevertheless, current candidate malaria vaccines do not elicit consistent, durable protection. Here we demonstrate that an alternative to conventional immunization, antibody gene delivery by a viral vector [vectored immunoprophylaxis (VIP)], can direct sustained expression of protective mAb in vivo and prevent P. falciparum infection in a rodent model. These studies provide a proof of principle for the use of VIP against the deadliest form of human malaria. Malaria caused by Plasmodium falciparum kills nearly one million children each year and imposes crippling economic burdens on families and nations worldwide. No licensed vaccine exists, but infection can be prevented by antibodies against the circumsporozoite protein (CSP), the major surface protein of sporozoites, the form of the parasite injected by mosquitoes. We have used vectored immunoprophylaxis (VIP), an adeno-associated virus-based technology, to introduce preformed antibody genes encoding anti-P. falciparum CSP mAb into mice. VIP vector-transduced mice exhibited long-lived mAb expression at up to 1,200 µg/mL in serum, and up to 70% were protected from both i.v. and mosquito bite challenge with transgenic Plasmodium berghei rodent sporozoites that incorporate the P. falciparum target of the mAb in their CSP. Serum antibody levels and protection from mosquito bite challenge were dependent on the dose of the VIP vector. All individual mice expressing CSP-specific mAb 2A10 at 1 mg/mL or more were completely protected, suggesting that in this model system, exceeding that threshold results in consistent sterile protection. Our results demonstrate the potential of VIP as a path toward the elusive goal of immunization against malaria.

Neonatal sex steroids affect responses to Seoul virus infection in male but not female Norway rats.

Previous studies illustrate that after inoculation with Seoul virus (i.e., the naturally occurring hantavirus found in Norway rats), adult male rats produce higher antibody responses, exhibit higher Th1 responses (i.e., IgG2a, IL-2, and IFN gamma), and shed virus longer than females, but these difference are not altered by manipulation of sex steroids in adulthood. To determine whether sex steroid hormones organize adult responses to hantavirus infection, at 2-4 days of age, male rats were gonadectomized and female rats were injected with testosterone. As adults, animals were inoculated with 10(4)pfu of Seoul virus. Neonatally gonadectomized males (NGM), control females (CF), and neonatal testosterone-treated females (NTF) had lower anti-Seoul virus IgG and IgG2a responses than control males (CM) 20, 30, and 40 days post-inoculation. Neonatal testosterone treatment had no effect on female antibody responses to infection. NGM, CF, and NTF shed virus in saliva and feces for a shorter duration than CM. There was no effect of neonatal hormone manipulation either on the percentage of animals with detectable virus or on the number of virus copies within each target organ. Genetic males, regardless of hormone manipulation, had higher virus replication in lung tissue than did genetic females. Neonatal sex steroids influence the sexual dimorphism in host immune function but do not modify virus replication in target tissues.