Defining functions for the L4-33K protein in adenovirus alternative splicing

The National Institute of Allergy and Infectious Diseases awarded the University of Pennsylvania $100,228 on January 9, 2027, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to define the functions of the L4-33K protein in adenovirus alternative splicing.

The award funds research to elucidate how the adenovirus L4-33K protein modulates viral alternative splicing and coordinates genome replication. The investigator proposes that L4-33K promotes the use of alternative, weak 3' splice sites across viral transcripts through direct interactions with cellular splicing factors and modulates viral DNA replication to prevent trans-complementation. Preliminary data show that L4-33K interacts with cellular proteins linked to RNA splicing, DNA replication, and repair, and localizes at replication forks where it is necessary for proper viral genome replication. In the absence of L4-33K, viral replication compartments enlarge and viral genome levels increase, indicating a role beyond splicing in influencing viral genome fate.

Work is performed in Philadelphia, Pennsylvania. The period of performance extends from January 9, 2027, through July 31, 2029. This is a project grant, the assistance type used for investigator-initiated biomedical research and research training in infectious diseases and related areas.

Abstract

As obligate intracellular pathogens, viruses have evolved diverse strategies to hijack the host's RNA processing machinery to promote viral splicing. Many viruses temporally coordinate genome replication with dramatic changes in transcription and alternative splicing [refs]. However, the mechanisms underlying this coordination, and the cellular proteins involved, remain poorly understood. Adenovirus (ADV) is a double-stranded DNA virus that replicates in the nucleus and encodes many overlapping transcriptional units, each carrying several spliced transcripts. I propose to utilize ADV as a molecular tool to explore the mechanisms viruses employ to coordinate alternative splicing with genome replication. The ADV L4-33 kilodalton protein (L4-33K) has been proposed to promote viral alternative splicing during the late stage of INFECTION7,8,9. Previous work has demonstrated a role of L4-33K in promoting the use of alternative, weak 3′ splice sites across viral TRANSCRIPTS7. However, the mechanisms by which L4-33K modulates ADV splicing are unknown. My preliminary data reveal that L4-33K interacts with many cellular proteins linked to RNA splicing as well as DNA replication and repair. Some of these RNA processing factors have been suggested to modulate U2 snrnp binding at 3′ splice sites, consistent with the known role of L4-33K in activating weak 3′ splice sites. Additionally, L4-33K is located at replication forks and is necessary for proper viral genome replication. In the absence of L4-33K, viral replication compartments enlarge, and viral genome levels are increased. Together, these results indicate that L4-33K plays an additional role beyond splicing, influencing viral genome replication and ultimately genome fate. I propose that L4-33K coordinates ADV alternative splicing via direct interactions with cellular 3′ splicing factors and modulates viral DNA replication to prevent transcription-replication conflicts (trcs). In aim 1, i will determine whether L4-33K can bind to viral or host RNA transcripts and which of the 3′ splice site factors are necessary for infection. I will determine whether L4-33K stimulates the pro-splicing activity of these 3′ splice site factors on viral transcripts. I will map the interacting domains within L4-33K and utilize a viral minigene splicing reporter as a readout for splicing activity. In aim 2, i will determine if L4-33K is present at viral or cellular replication forks and whether L4-33K is able to slow viral genome replication to prevent trcs. Additionally, i will explore interactions between L4-33K and host DNA replication and repair proteins in preventing viral trcs. The results from this aim will elucidate mechanisms by which L4-33K regulates viral splicing and reveal a novel link between L4-33K and TRC resolution. Furthermore, this will be one of the first studies demonstrating a connection between a viral splicing regulator and trcs. This work will have broad implications for the fields of virology and alternative splicing, as well as emerging research areas investigating the interplay between DNA replication and transcription. This work will be conducted in the weitzman lab at the children's hospital of philadelphia, where i will receive interdisciplinary training to support my future research in alternative splicing and viral infection.