Tailed bacteriophages use a DNA-packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component of this DNA-packaging machinery acts as a molecular matchmaker that recognizes both the viral genome and the main motor component, the large terminase (TerL). However, how TerS binds DNA and the TerL protein remains unclear. Here, we identified gp83 of the thermophilic bacteriophage P74-26 as the TerS protein. We found that TerS(P76-26) oligomerizes into a nonamer that binds DNA, stimulates TerL ATPase activity, and inhibits TerL nuclease activity. A cryo-EM structure of TerS(P76-26) revealed that it forms a ring with a wide central pore and radially arrayed helix-turn-helix (HTH) domains. The structure further showed that these HTH domains, which are thought to bind DNA by wrapping the double helix around the ring, are rigidly held in an orientation distinct from that seen in other TerS proteins. This rigid arrangement of the putative DNA-binding domain imposed strong constraints on how TerS(P76-26) can bind DNA. Finally, the TerS(P76-26) structure lacked the conserved C-terminal beta-barrel domain used by other TerS proteins for binding TerL. This suggests that a well-ordered C-terminal beta-barrel domain is not required for TerS(P76-26) to carry out its matchmaking function. Our work highlights a thermophilic system for studying the role of small terminase proteins in viral maturation and presents the structure of TerS(P76-26), revealing key differences between this thermophilic phage and its mesophilic counterparts.

The SARS-CoV-2 spike (S) protein variant D614G supplanted the ancestral virus worldwide, reaching near fixation in a matter of months. Here we show that D614G was more infectious than the ancestral form on human lung cells, colon cells, and on cells rendered permissive by ectopic expression of human ACE2 or of ACE2 orthologs from various mammals, including Chinese rufous horseshoe bat and Malayan pangolin. D614G did not alter S protein synthesis, processing, or incorporation into SARS-CoV-2 particles, but D614G affinity for ACE2 was reduced due to a faster dissociation rate. Assessment of the S protein trimer by cryo-electron microscopy showed that D614G disrupts an interprotomer contact and that the conformation is shifted toward an ACE2 binding-competent state, which is modeled to be on pathway for virion membrane fusion with target cells. Consistent with this more open conformation, neutralization potency of antibodies targeting the S protein receptor-binding domain was not attenuated.