Alphaviruses are mosquito-borne viruses that cause serious disease in humans and other mammals. Along with its mosquito vector, the Alphavirus chikungunya virus (CHIKV) has spread explosively in the last 20 years, and there is no approved treatment for chikungunya fever. On the plasma membrane of the infected cell, CHIKV generates dedicated organelles for viral RNA replication, so-called spherules. Whereas structures exist for several viral proteins that make up the spherule, the architecture of the full organelle is unknown. Here, we use cryo-electron tomography to image CHIKV spherules in their cellular context. This reveals that the viral protein nsP1 serves as a base for the assembly of a larger protein complex at the neck of the membrane bud. Biochemical assays show that the viral helicase-protease nsP2, while having no membrane affinity on its own, is recruited to membranes by nsP1. The tomograms further reveal that full-sized spherules contain a single copy of the viral genome in double-stranded form. Finally, we present a mathematical model that explains the membrane remodeling of the spherule in terms of the pressure exerted on the membrane by the polymerizing RNA, which provides a good agreement with the experimental data. The energy released by RNA polymerization is found to be sufficient to remodel the membrane to the characteristic spherule shape.
Replication forks often stall at damaged DNA. Resumption of DNA synthesis can occur by replacement of the replicative DNA polymerase with specialized, error-prone translesion DNA polymerases (TLS), that have higher tolerance for damaged substrates. Several of these polymerases (Polλ, Polη and PrimPol) are stimulated in DNA synthesis through interaction with PolDIP2, however the mechanism of this PolDIP2-dependent stimulation is still unclear. Here we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPol's enhanced processivity. PolDIP2 increases PrimPol's primer-template and dNTP binding affinity, which concomitantly enhances PrimPol's nucleotide incorporation efficiency. This activity is dependent on a unique arginine cluster in PolDIP2 and could be essential for PrimPol to function in vivo, since the polymerase activity of PrimPol alone is very limited. This mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, could be common to all other PolDIP2interacting TLS polymerases, i.e. Polλ, Polη, Polζ and REV1, and might be critical for their in vivo function of tolerating DNA lesions at physiological nucleotide concentrations.replication fork progression (4-6). Among various TLS polymerases, the primase/polymerase PrimPol has been shown to help both in nuclear and mitochondrial DNA replication fork progression (7-8). Additionally, PrimPol can also re-prime downstream of blocking lesions, thus re-reinitiating DNA replication (1,(9)(10)(11)(12)(13)(14)(15).PrimPol is a monomeric enzyme belonging to the archaeal-eukaryotic primase (AEP) superfamily (16). Its catalytic core contains three highly conserved motifs (see Figure1A) which build the dNTP binding site used for elongation (17). To start primer synthesis, PrimPol requires a unique zinc finger (ZnF)-containing C-terminal domain, which facilitates binding of the first 5´-nucleotide (1, 18). Beside its function as a primase, PrimPol behaves in vitro as a polymerase with very low processivity (9, 19), suggesting the need for a cofactor to function optimally in the cell. In contrast to some other TLS polymerases (20), PrimPol's activity is not regulated by the sliding clamp Proliferating Cell Nuclear Antigen (PCNA) (21), suggesting that another factor might control its function. Indeed, polymerase d-interacting protein 2 (PolDIP2; also known as PDIP38) and single stranded DNA binding protein Replication Protein A (RPA) (22-24) are able to stimulate PrimPol-dependent DNA synthesis. Interestingly, PolDIP2was previously shown to physically interact with TLS polymerases Polz and REV1 (25), as well as to increase the processivity of Poll and Polh (26,27). Therefore, many TLS DNA polymerases could be regulated by a common, still to be elucidated, PolDIP2dependent mechanism.Here we show that PrimPol uses a flexible loop located at its AEP catalytic core to interact with the conserved ApaG-like C-terminal region of PolDIP2 (28), which is essential for the stimul...
Alphaviruses are mosquito-borne viruses that cause serious disease in humans and other mammals. Along with its mosquito vector, the alphavirus chikungunya virus (CHIKV) has spread explosively in the last 20 years, and there is no approved treatment for chikungunya fever. On the plasma membrane of the infected cell, CHIKV generates dedicated organelles for viral RNA replication, so-called spherules. Whereas structures exist for several viral proteins that make up the spherule, the architecture of the full organelle is unknown. Here, we use cryo-electron tomography to image CHIKV spherules in their cellular context. This reveals that the viral protein nsP1 serves as a base for the assembly of a larger protein complex at the neck of the membrane bud. Biochemical assays show that the viral helicase-protease nsP2, while having no membrane affinity on its own, is recruited to membranes by nsP1. The tomograms further reveal that full-sized spherules contain a single copy of the viral genome in double-stranded form. Finally, we present a mathematical model that explains the membrane remodeling of the spherule in terms of the pressure exerted on the membrane by the polymerizing RNA, which provides a good agreement with the experimental data. The energy released by RNA polymerization is found to be sufficient to remodel the membrane to the characteristic spherule shape.
Replication forks often stall at damaged DNA. To overcome these obstructions and complete the DNA duplication in a timely fashion, replication can be restarted downstream of the DNA lesion. In mammalian cells, this repriming of replication can be achieved through the activities of primase and polymerase PrimPol. PrimPol is stimulated in DNA synthesis through interaction with PolDIP2, however the exact mechanism of this PolDIP2-dependent stimulation is still unclear. Here, we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPol's enhanced processivity. PolDIP2 increases primer-template and dNTP binding affinities of PrimPol, which concomitantly enhances its nucleotide incorporation efficiency. This stimulation is dependent on a unique arginine cluster in PolDIP2. Since the polymerase activity of PrimPol alone is very limited, this mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, might be critical for the in vivo function of PrimPol in tolerating DNA lesions at physiological nucleotide concentrations.
In this study, we discover the tetraspanin CD81 as a host factor for the globally emerging chikungunya virus and related alphaviruses. We show that CD81 promotes replication of viral genomes in human and mouse cells, while virus entry into cells is independent of CD81.
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