Conserved cysteine residues in Kaposi's sarcoma herpesvirus ORF34 are necessary for viral production and viral pre-initiation complex formation.

Kaposi's sarcoma herpesvirus (KSHV) ORF34 plays a significant role as a component of the viral pre-initiation complex (vPIC), which is indispensable for late gene expression across beta- and gammaherpesviruses. Although the key role of ORF34 within the vPIC and its function as a hub protein have been recognized, further clarification regarding its specific contribution to vPIC functionality and interactions with other components is required. This study employed a deep learning algorithm-assisted structural model of ORF34, revealing highly conserved amino acid residues across human beta- and gammaherpesviruses localized in structured domains. Thus, we engineered ORF34 alanine-scanning mutants by substituting conserved residues with alanine. These mutants were evaluated for their ability to interact with other vPIC factors and restore viral production in cells harboring the ORF34-deficient KSHV-BAC. Our experimental results highlight the crucial role of the four cysteine residues conserved in ORF34: a tetrahedral arrangement consisting of a pair of C-Xn-C consensus motifs. This suggests the potential incorporation of metal cations in interacting with ORF24 and ORF66 vPIC components, facilitating late gene transcription, and promoting overall virus production by capturing metal cations. In summary, our findings underline the essential role of conserved cysteines in KSHV ORF34 for effective vPIC assembly and viral replication, thereby enhancing our understanding of the complex interplay between the vPIC components. IMPORTANCE: The initiation of late gene transcription is universally conserved across the beta- and gammaherpesvirus families. This process employs a viral pre-initiation complex (vPIC), which is analogous to a cellular PIC. Although KSHV ORF34 is a critical factor for viral replication and is a component of the vPIC, the specifics of vPIC formation and the essential domains crucial for its function remain unclear. Structural predictions suggest that the four conserved cysteines (C170, C175, C256, and C259) form a tetrahedron that coordinates the metal cation. We investigated the role of these conserved amino acids in interactions with other vPIC components, late gene expression, and virus production to demonstrate for the first time that these cysteines are pivotal for such functions. This discovery not only deepens our comprehensive understanding of ORF34 and vPIC dynamics but also lays the groundwork for more detailed studies on herpesvirus replication mechanisms in future research.

Evaluating HIV-1 Infectivity and Virion Maturation across Varied Producer Cells with a Novel FRET-Based Detection and Quantification Assay.

The maturation of HIV-1 virions is a crucial process in viral replication. Although T-cells are a primary source of virus production, much of our understanding of virion maturation comes from studies using the HEK293T human embryonic kidney cell line. Notably, there is a lack of comparative analyses between T-cells and HEK293T cells in terms of virion maturation efficiency in existing literature. We previously developed an advanced virion visualization system based on the FRET principle, enabling the effective distinction between immature and mature virions via fluorescence microscopy. In this study, we utilized pseudotyped, single-round infectious viruses tagged with FRET labels (HIV-1 Gag-iFRET∆Env) derived from Jurkat (a human T-lymphocyte cell line) and HEK293T cells to evaluate their virion maturation rates. HEK293T-derived virions demonstrated a maturity rate of 81.79%, consistent with other studies and our previous findings. However, virions originating from Jurkat cells demonstrated a significantly reduced maturation rate of 68.67% (p < 0.0001). Correspondingly, viruses produced from Jurkat cells exhibited significantly reduced infectivity compared to those derived from HEK293T cells, with the relative infectivity measured at 65.3%. This finding is consistent with the observed relative maturation rate of viruses produced by Jurkat cells. These findings suggest that initiation of virion maturation directly correlates with viral infectivity. Our observation highlights the dynamic nature of virus-host interactions and their implications for virion production and infectivity.

Evaluating HIV-1 Infectivity and Virion Maturation Across Varied Producer Cells with a Novel FRET-Based Detection and Quantification Assay.

The maturation of HIV-1 virions is a crucial process in viral replication. Although T cells are a primary source of virus production, much of our understanding of virion maturation comes from studies using the HEK293T human embryonic kidney cell line. Notably, there is a lack of comparative analyses between T cells and HEK293T cells in terms of virion maturation efficiency in existing literature. We previously developed an advanced virion visualization system based on the FRET principle, enabling the effective distinction between immature and mature virions via fluorescence microscopy. In this study, we utilized pseudotyped, single-round infectious viruses tagged with FRET labels (HIV-1 Gag-iFRETΔEnv) derived from Jurkat (a human T lymphocyte cell line) and HEK293T cells to evaluate their virion maturation rates. HEK293T-derived virions demonstrated a maturity rate of 81.79%, consistent with other studies and our previous findings. However, virions originating from Jurkat cells demonstrated a significantly reduced maturation rate of 68.67% (p < 0.0001). Correspondingly, viruses produced from Jurkat cells exhibited significantly reduced infectivity compared to those derived from HEK293T cells, with the relative infectivity measured at 65.3%. This finding is consistent with the observed relative maturation rate of viruses produced by Jurkat cells. These findings suggest that initiation of virion maturation directly correlates with viral infectivity. Our observation highlights the dynamic nature of virus-host interactions and their implications for virion production and infectivity.