Abstract
Ancient retroviral infections and integrations of the reverse transcribed viral DNA into the genomes of germline cells gave rise to endogenous retroviruses, which now comprise about 8% of the human genome. Human endogenous retrovirus K (HERV-K, HML-2) represents the most recently integrated and transcriptionally active endogenous retrovirus family in the human genome. While most HERV elements are highly mutated and suppressed, several HERV-K (HML-2) loci retain open reading frames capable of producing viral RNAs and proteins, and aberrant expression of HERV-K has been associated with various malignancies.
The HERV-K (HML-2) accessory protein Rec, a functional homolog of HIV-1 Rev, mediates nuclear export of unspliced viral RNAs through the Rec Response Element (RcRE), enabling expression of structural proteins. However, the functional diversity of Rec proteins encoded across genomic HERV-K (HML-2) loci and their potential roles in human disease remain poorly understood.
This dissertation documents the results of systematically characterizing the post-transcriptional functions of the Rec proteins encoded by all the annotated HERV-K (HML-2) proviral loci and investigates the oncogenic potential of the most common functional variant (named HERV-K Con Rec). We identified 28 loci predicted to encode intact Rec proteins and functionally tested them using a dual-color RcRE fluorescent reporter assay as well as an ELISA assay that measures HIV p24 expression from an mRNA with a retained intron. Nine loci were shown to encode functional Rec proteins capable of promoting RcRE-dependent post-transcriptional expression, with most being insertionally polymorphic, suggesting ongoing evolutionary dynamics. Several Rec variants exhibited trans-dominant negative activity, inhibiting functional Rec proteins when co-expressed. The most potent trans-dominant negative Rec encoded by provirus 12q14.1 differs from the HERV-K Con Rec at only 2 positions, with a change from asparagine to histidine at position 8 and deletion of glutamic acid at position 34 (ΔE34). Mutational analysis revealed that the deletion of glutamic acid in HERV-K Con Rec is sufficient for full trans-dominant negative activity.
To assess the oncogenic potential of HERV-K Con Rec, this protein was expressed in non-tumorigenic MCF-10A breast epithelial cells alone or in combination with oncogenic HRASG12V. While Rec alone did not transform cells, co-expression significantly enhanced anchorage-independent growth and colony formation induced by the HRAS mutant. Co-culture experiments revealed paracrine effects, with HRASG12V+ Rec-expressing cells promoting larger colony formation of neighboring HRASG12V-only cells. In nude mice xenograft models, Rec co-expression with HRASG12V increased tumor incidence, size, and progression, with histopathological evidence of stromal remodeling and micrometastasis. Transcriptomic profiling revealed the upregulation of extracellular matrix remodeling genes and suppression of interferon-stimulated genes, consistent with immune evasion and tumor-promoting microenvironmental changes.
Together, these studies define the functional diversity among the Rec proteins encoded in the human genome and demonstrate that HERV-K (HML-2) Rec cooperates with oncogenic HRAS to promote tumorigenesis through both cell-intrinsic and microenvironmental mechanisms. These findings expand our understanding of HERV-K (HML-2) post-transcriptional regulation and suggest that Rec contributes to the progression of cancers with reactivated HML-2 expression.
