The rubella virus (RUB) capsid protein (C) is a multifunctional phosphoprotein with roles beyond encapsidation. It is able to rescue a large lethal deletion of the Q domain in the P150 replicase gene at a step in replication before detectable viral RNA synthesis, indicating a common function shared by RUB C and the Q domain. The goal of this dissertation was to use constructs containing the N-terminal 88 amino acids of RUB C, the region previously defined as the minimal region required for the rescue of Q domain mutants, to elucidate the function of RUB C in Q domain rescue and viral RNA synthesis. In the first specific aim, the rescue function of 1-88 RUB C and the importance of an arginine-rich cluster, R2, within 1-88 RUB C for rescue were confirmed. Rescue was not correlated with intracellular localization or phosphorylation status of RUB C. In the second specific aim, the involvement of RUB C in early events post-transfection with RUB RNA was analyzed. RUB C specifically protected RUB transcripts early post-transfection and protection required R2. However, it was concluded the protection observed was due to the encapsidation function of RUB C and not related to Q domain rescue. No differences in the translation of the RUB nonstructural proteins in the presence or absence of RUB C were observed. Interactions of RUB C with host cell proteins were analyzed. Although the interaction of RUB C with cellular p32 required the R2 cluster, both wild type (does not require RUB C for replication) and RQQ (requires RUB C for replication) Q domain bound p32, indicating interaction with this binding partner is not the basis of rescue. Using a human protein array phosphatidylinositol transfer protein alpha isoform (PITPα) was found to interact with RUB C but not its R2 mutant. However, co-immunoprecipitation experiments revealed that this protein binds both forms of RUB C. Although the mechanism behind the rescue of the RUB P150 Q domain by RUB C remains unknown, we propose a model that RUB C plays a role in generation of the virus replication complex in infected cells.

Rubella virus (RUBV) is a positive-strand RNA virus and the causative agent of rubella and congenital rubella syndrome in humans. To replicate its RNA, RUBV forms membrane-associated spherules, called replication complexes (RCs), the induction of which requires the two virus nonstructural proteins (NSPs), P150 and P90. Interestingly, late in infection the NSPs form a unique cytoplasmic fiber network, similar in appearance to microtubules, the function of which is unknown. Little is known about the roles of the RUBV NSPs in forming these structures and, to this end, we scrutinized the behavior and biochemical properties of the NSPs, both after expression from plasmids and during RUBV infection, using mutagenic, biochemical and pharmacological approaches. The following findings were made: First, the precursor from which P150 and P90 are produced via an embedded protease at the C-terminus of P150, called P200, was required for initial targeting to cytoplasmic foci. P150 was the determinant of fiber formation and while P90 had no specific targeting sequences on its own, P90 sequences within P200 were required for correct targeting of P200. An alpha-helix at the N-terminus of P150 was also important for correct targeting of P200, putatively by mediating the interaction between P150 and P90 within the precursor. Second, the membrane binding domain within the NSPs was within the N-terminal ~450 amino acids of P150. P150 is in an exceptionally tight association with membranes. Third, both the N- and C-terminal regions of P150, and specifically long alpha-helices within these regions, are necessary for fiber formation. Fiber formation relied on an intact microtubule network, but neither microtubule repositioning nor dynamic movement along microtubules was required. Additionally, it was shown that microtubules were not necessary in RUBV replication. Finally, P150 fibers were not required for RUBV replication; however, it was shown that the fibers are likely important in formation of cytoplasmic extensions through which a novel system of cell-to-cell transport of viral RNA in the absence of virus particles appears to occur.

The rubella virus (RV) was the first virus shown to be teratogenic in humans. The wealth of data on the clinical symptoms associated with congenital rubella syndrome is in stark contrast to an incomplete understanding of the forces leading to the teratogenic alterations in humans. This applies not only to RV, but also to congenital viral infections in general and includes (1) the mode of vertical transmission, even at early gestation, (2) the possible involvement of inflammation as a consequence of an activated innate immune response, and (3) the underlying molecular and cellular alterations. With the progress made in the development of pluripotent stem cell-based models including organoids and embryoids, it is now possible to assess congenital virus infections on a mechanistic level. Moreover, antiviral treatment options can be validated, and newly emerging viruses with a potential impact on human embryonal development, such as that recently reflected by the Zika virus (ZIKV), can be characterized. Here, we discuss human cytomegalovirus (HCMV) and ZIKV in comparison to RV as viruses with well-known congenital pathologies and highlight their analysis on current models for the early phase of human development. This includes the implications of their genetic variability and, as such, virus strain-specific properties for their use as archetype models for congenital virus infections. In this review, we will discuss the use of induced pluripotent stem cells (iPSC) and derived organoid systems for the study of congenital virus infections with a focus on their prominent aetiologies, HCMV, ZIKV, and RV. Their assessment on these models will provide valuable information on how human development is impaired by virus infections; it will also add new insights into the normal progression of human development through the analysis of developmental pathways in the context of virus-induced alterations. These are exciting perspectives for both developmental biology and congenital virology.