Michael Nevels

“Hit-and-Run” Transformation by Adenovirus Oncogenes

ABSTRACT According to classical concepts of viral oncogenesis, the persistence of virus-specific oncogenes is required to maintain the transformed cellular phenotype. In contrast, the “hit-and-run” hypothesis claims that viruses can mediate cellular transformation through an initial “hit,” while maintenance of the transformed state is compatible with the loss (“run”) of viral molecules. It is well established that the adenovirus E1A and E1B gene products can cooperatively transform primary human and rodent cells to a tumorigenic phenotype and that these cells permanently express the viral oncogenes. Additionally, recent studies have shown that the adenovirus E4 region encodes two novel oncoproteins, the products of E4orf6 and E4orf3, which cooperate with the viral E1A proteins to transform primary rat cells in an E1B-like fashion. Unexpectedly, however, cells transformed by E1A and either E4orf6 or E4orf3 fail to express the viral E4 gene products, and only a subset contain E1A proteins. In fact, the majority of these cells lack E4- and E1A-specific DNA sequences, indicating that transformation occurred through a hit-and-run mechanism. We provide evidence that the unusual transforming activities of the adenoviral oncoproteins may be due to their mutagenic potential. Our results strongly support the possibility that even tumors that lack any detectable virus-specific molecules can be of viral origin, which could have a significant impact on the use of adenoviral vectors for gene therapy.

Temporal Dynamics of Cytomegalovirus Chromatin Assembly in Productively Infected Human Cells

ABSTRACT The genomes of herpesviruses, including human cytomegalovirus (CMV), are double-stranded DNA molecules maintained as episomes during infection. The viral DNA lacks histones when encapsidated in the virion. However, it has been found histone associated inside infected cells, implying unidentified chromatin assembly mechanisms. Our results indicate that components of the host cell nucleosome deposition machinery target intranuclear CMV DNA, resulting in stepwise viral-chromatin assembly. CMV genomes undergo limited histone association and nucleosome assembly as early as 30 min after infection via DNA replication-independent mechanisms. Low average viral-genome chromatinization is maintained throughout the early stages of infection. The late phase of infection is characterized by a striking increase in average histone occupancy coupled with the process of viral-DNA replication. While the initial chromatinization affected all analyzed parts of the CMV chromosome, a subset of viral genomic regions, including the major immediate-early promoter, proved to be largely resistant to replication-dependent histone deposition. Finally, our results predict the likely requirement for an unanticipated chromatin disassembly process that enables packaging of histone-free DNA into progeny capsids.

Snapshots: Chromatin Control of Viral Infection

Like their cellular host counterparts, many invading viral pathogens must contend with, modulate, and utilize the host cells chromatin machinery to promote efficient lytic infection or control persistent-latent states. While not intended to be comprehensive, this review represents a compilation of conceptual snapshots of the dynamic interplay of viruses with the chromatin environment. Contributions focus on chromatin dynamics during infection, viral circumvention of cellular chromatin repression, chromatin organization of large DNA viruses, tethering and persistence, viral interactions with cellular chromatin modulation machinery, and control of viral latency-reactivation cycles.

SUMOylation of the Human Cytomegalovirus 72-Kilodalton IE1 Protein Facilitates Expression of the 86-Kilodalton IE2 Protein and Promotes Viral Replication

ABSTRACT The 72-kDa immediate-early 1 protein (IE1-72kDa) of human cytomegalovirus has been previously shown to be posttranslationally modified by covalent conjugation to the ubiquitin-related protein SUMO-1. Using an infectious bacterial artificial chromosome clone of human cytomegalovirus, we constructed a mutant virus (BADpmIE1-K450R) that is deficient for SUMOylation of IE1-72kDa due to a single amino acid exchange in the SUMO-1 attachment site. Compared to wild-type virus, this mutant grew more slowly and generated a reduced yield in infected human fibroblasts, indicating that SUMO modification is required for the full activity of IE1-72kDa. The lack of SUMOylation did not affect the intranuclear localization of IE1-72kDa, including its ability to target to and disrupt PML bodies and to bind to mitotic chromatin. Likewise, SUMOylation-deficient IE1-72kDa activated several viral promoters as efficiently as the wild-type protein. However, the failure to modify IE1-72kDa resulted in substantially reduced levels of the IE2 transcript and its 86-kDa protein (IE2-86kDa). These observations suggest that SUMO modification of IE1-72kDa contributes to efficient HCMV replication by promoting the accumulation of IE2-86kDa.

Murine Cytomegalovirus m41 Open Reading Frame Encodes a Golgi-Localized Antiapoptotic Protein

ABSTRACT Viruses have evolved various strategies to prevent premature apoptosis of infected host cells. Some of the viral genes mediating antiapoptotic functions have been identified by their homology to cellular genes, but others are structurally unrelated to genes of known function. In this study, we used a random, unbiased approach to identify such genes in the murine cytomegalovirus genome. From a library of random transposon insertion mutants, a mutant virus that caused premature cell death was isolated. The transposon was inserted within open reading frame m41. An independently constructed m41 deletion mutant showed the same phenotype, whereas deletion mutants lacking the adjacent genes m40 and M42 did not. Apoptosis occurred in different cell types, could be blocked by caspase inhibitors, and did not require p53. Within the murine cytomegalovirus genome, m41, m40, and m39 form a small cluster of genes of unknown function. They are homologous to r41, r40, and r39 of rat cytomegalovirus, but lack sequence homology to UL41, UL40, and UL37 exon 1 (UL37x1) which are located at the corresponding positions of the human cytomegalovirus genome. Unlike UL37x1 of human cytomegalovirus, which encodes a mitochondrion-localized inhibitor of apoptosis that is essential for virus replication, m41 encodes a protein that localizes to the Golgi apparatus. The murine cytomegalovirus m41 product is the first example of a Golgi-localized protein that prevents premature apoptosis and thus extends the life span of infected cells.

Two Distinct Activities Contribute to the Oncogenic Potential of the Adenovirus Type 5 E4orf6 Protein

ABSTRACT Previous studies have shown that the adenovirus type 5 (Ad5) E4orf6 gene product displays features of a viral oncoprotein. It initiates focal transformation of primary rat cells in cooperation with Ad5 E1 genes and confers multiple additional transformed properties on E1-expressing cells, including profound morphological alterations and dramatically accelerated tumor growth in nude mice. It has been reported that E4orf6 binds to p53 and, in the presence of the Ad5 E1B-55kDa protein, antagonizes p53 stability by targeting the tumor suppressor protein for active degradation. In the present study, we performed a comprehensive mutant analysis to assign transforming functions of E4orf6 to distinct regions within the viral polypeptide and to analyze a possible correlation between E4orf6-dependent p53 degradation and oncogenesis. Our results show that p53 destabilization maps to multiple regions within both amino- and carboxy-terminal parts of the viral protein and widely cosegregates with E4orf6-dependent acceleration of tumor growth, indicating that both effects are related. In contrast, promotion of focus formation and morphological transformation require only a carboxy-terminal segment of the E4 protein. Thus, these effects are completely independent of p53 stability, but may involve other interactions with the tumor suppressor. Our results demonstrate that at least two distinct activities contribute to the oncogenic potential of Ad5 E4orf6. Although genetically separable, both activities are largely mediated through a novel highly conserved, cysteine-rich motif and a recently described arginine-faced amphipathic alpha helix, which resides within a carboxy-terminal “oncodomain” of the viral protein.

The adenovirus E4orf6 protein contributes to malignant transformation by antagonizing E1A-induced accumulation of the tumor suppressor protein p53

The adenovirus type 5 (Ad5) E4orf6 protein promotes focus formation of primary baby rat kidney (BRK) cells in cooperation with Ad5 E1 proteins. This activity is most likely related to the ability of the E4orf6 protein to bind to p53 and modulate its tumor suppressor functions. In this study we report that transformed BRK cells that stably express E4orf6 in addition to E1A and E1B (ABS cells) displayed multiple additional properties commonly associated with a high grade of oncogenic transformation compared to cells expressing only E1A and E1B (AB cells). These properties included morphological alterations, markedly enhanced growth rates and growth to much higher saturation densities. Following injection into nude mice ABS-derived tumors exhibited accelerated growth and, based on histopathological criteria, proofed to be much more malignant compared to tumors generated by AB cells. Interestingly, these highly transformed properties of ABS cells correlated with a dramatic reduction of p53 steady-state levels which inversely correlated with E4orf6 expression. From these results we conclude that expression of the Ad5 E4orf6 protein (i) confers additional transformed in vitro properties to primary rat cells expressing the Ad5 E1 proteins, and (ii) increases the tumorigenic and malignant potential of these cells in vivo. Our data suggest that the Ad5 E4orf6 protein enhances the intrinsic ability of E1-transformed rat cells to grow in a neoplastic state by completely inactivating p53 tumor suppressor function in combination with the E1A and E1B proteins.

Physical Requirements and Functional Consequences of Complex Formation between the Cytomegalovirus IE1 Protein and Human STAT2

ABSTRACT Our previous work has shown that efficient evasion from type I interferon responses by human cytomegalovirus (hCMV) requires expression of the 72-kDa immediate-early 1 (IE1) protein. It has been suggested that IE1 inhibits interferon signaling through intranuclear sequestration of the signal transducer and activator of transcription 2 (STAT2) protein. Here we show that physical association and subnuclear colocalization of IE1 and STAT2 depend on short acidic and serine/proline-rich low-complexity motifs in the carboxy-terminal region of the 491-amino-acid viral polypeptide. These motifs compose an essential core (amino acids 373 to 420) and an adjacent ancillary site (amino acids 421 to 445) for STAT2 interaction that are predicted to form part of a natively unstructured domain. The presence of presumably “disordered” carboxy-terminal domains enriched in low-complexity motifs is evolutionarily highly conserved across all examined mammalian IE1 orthologs, and the murine cytomegalovirus IE1 protein appears to interact with STAT2 just like the human counterpart. A recombinant hCMV specifically mutated in the IE1 core STAT2 binding site displays hypersensitivity to alpha interferon, delayed early viral protein accumulation, and attenuated growth in fibroblasts. However, replication of this mutant virus is specifically restored by knockdown of STAT2 expression. Interestingly, complex formation with STAT2 proved to be entirely separable from disruption of nuclear domain 10 (ND10), another key activity of IE1. Finally, our results demonstrate that IE1 counteracts the antiviral interferon response and promotes viral replication by at least two distinct mechanisms, one depending on sequestration of STAT2 and the other one likely involving ND10 interaction.

Human Cytomegalovirus IE1 Protein Elicits a Type II Interferon-Like Host Cell Response That Depends on Activated STAT1 but Not Interferon-γ

Human cytomegalovirus (hCMV) is a highly prevalent pathogen that, upon primary infection, establishes life-long persistence in all infected individuals. Acute hCMV infections cause a variety of diseases in humans with developmental or acquired immune deficits. In addition, persistent hCMV infection may contribute to various chronic disease conditions even in immunologically normal people. The pathogenesis of hCMV disease has been frequently linked to inflammatory host immune responses triggered by virus-infected cells. Moreover, hCMV infection activates numerous host genes many of which encode pro-inflammatory proteins. However, little is known about the relative contributions of individual viral gene products to these changes in cellular transcription. We systematically analyzed the effects of the hCMV 72-kDa immediate-early 1 (IE1) protein, a major transcriptional activator and antagonist of type I interferon (IFN) signaling, on the human transcriptome. Following expression under conditions closely mimicking the situation during productive infection, IE1 elicits a global type II IFN-like host cell response. This response is dominated by the selective up-regulation of immune stimulatory genes normally controlled by IFN-γ and includes the synthesis and secretion of pro-inflammatory chemokines. IE1-mediated induction of IFN-stimulated genes strictly depends on tyrosine-phosphorylated signal transducer and activator of transcription 1 (STAT1) and correlates with the nuclear accumulation and sequence-specific binding of STAT1 to IFN-γ-responsive promoters. However, neither synthesis nor secretion of IFN-γ or other IFNs seems to be required for the IE1-dependent effects on cellular gene expression. Our results demonstrate that a single hCMV protein can trigger a pro-inflammatory host transcriptional response via an unexpected STAT1-dependent but IFN-independent mechanism and identify IE1 as a candidate determinant of hCMV pathogenicity.

The Human Cytomegalovirus Major Immediate-Early Proteins as Antagonists of Intrinsic and Innate Antiviral Host Responses

The major immediate-early (IE) gene of human cytomegalovirus (CMV) is believed to have a decisive role in acute infection and its activity is an important indicator of viral reactivation from latency. Although a variety of gene products are expressed from this region, the 72-kDa IE1 and the 86-kDa IE2 nuclear phosphoproteins are the most abundant and important. Both proteins have long been recognized as promiscuous transcriptional regulators. More recently, a critical role of the IE1 and IE2 proteins in counteracting non-adaptive host cell defense mechanisms has been revealed. In this review we will briefly summarize the available literature on IE1- and IE2-dependent mechanisms contributing to CMV evasion from intrinsic and innate immune responses.