The replication initiator protein (Rep) encoded by bacteriophages and circular plasmids is associated with the rolling-circle replication. An in vitro study of Rep encoded by the archaeal rudivirus suggested its function in recognition and cleavage of the viral terminal sequences. To further characterize this protein and its role in virus propagation, we performed in vivo studies of the Rep protein (gp16) from Sulfolobus islandicus rod-shaped virus 2 (SIRV2). Previously, a CRISPR-based genome editing method has been used to knock out accessory genes of SIRV2, generating seven mutant viruses (SIRV2M-SIRVMVII). Based on SIRV2MVII, the gp16 gene was knocked out. The loss of Rep caused a decrease in virus yield by 4-6 fold upon infection of Sulfolobus host lacking CRISPR arrays. Viruses with Rep have a higher number of intracellular virus genome copy than the mutant viruses, especially at 2 hpi by which the maximum difference reached 5-6-fold in the terminal region of the genome. With the Rep protein, the replication initiation at SIRV2 genome termini peaked between 1 hpi and 2 hpi. These results suggest that Rep is related to the replication initiation at the termini of the SIRV2 genome. Besides, we observed that viruses without Rep can still propagate, suggesting the existence of a second replication origin that mutant viruses must have used as an alternative replication initiation site. Thus, we sequenced the SIRV2 genome and found two internal regions with high coverage of mapped reads that could likely serve as replication origins. In a natural background, where hosts have CRISPR-Cas and viruses have anti-CRISPR proteins (Acrs), Rep produced >50 fold difference in virus yield, the maximum difference reaching ~180 fold, which is significantly higher than the fold of difference in the infected host lacking CRISPR immune systems. These results showed how Rep is crucial for efficient virus propagation in the virus-host competition, especially in the presence of CRISPR-Cas systems. Co-infection experiments showed how viruses with Rep (with all Acrs) outcompeted mutant virus (with all Acrs) upon infection of Sulfolobus strains with full functional CRISPR-Cas or individual CRISPR-Cas systems, indicating the Rep provides fitness advantage in virus-virus conflicts. 22 homologs of the Rep protein are identified in members of Rudiviridae, but this protein family is absent among Lipothrixviridae. A phylogenetic tree based on Rep grouped the rudviruses into distinct branches according to their geographical location of isolation. In the second project, we investigated the protein interactions and assembly of the spindle-shaped virus Sulfolobus monocaudavirus 1 (SMV1) particle. Previous studies showed SMV1 as a potential nanoplatform owing to its unique properties: thermo-stability and non-pathogenicity. To further develop SMV1 as a nano-vector, i.e. an empty capsid into which desired genetic elements can be packaged, the essential step is to understand the pathway of virus particle assembly. First, protein pull-down of 11 SMV1 virion proteins was performed, revealing that a small protein gp32 of unknown function can interact with both major coat proteins (gp06 and gp11). In addition, gp50, an RHH domain protein, is likely to form protein aggregates, the function of which remains to be characterized. We attempted on virus assembly by mixing all cell lysates containing expressed SMV1 virion proteins, followed by treatments with heat (78°C) and PEG. Long tube-like structures with sizes around 100 nm were observed by electron microscopy, which is smaller than the normal SMV1 virus particle size (around 200 nm). The irregular structures observed are probably due to incomplete protein interactions. The preliminary study presented here will help us to improve the experimental conditions for virus assembly in future work.