HYDRA, having discovered that Howard Stark had created five additional replicas of the Super Soldier Serum, similar to the ones that enhanced the abilities of Johann Schmidt and Steve Rogers, defrosted Bucky Barnes from the Cryostasis Chamber. While undergoing mind altercations at the HYDRA Siberian Facility, Vasily Karpov debriefed the assassin about his new mission and dispatched him to Long Island to assassinate the Starks. The Winter Soldier executed the Starks, staging their deaths as a car accident before retrieving the serum. Five members of HYDRA's most elite death squad were transfused with the serum, with HYDRA's intent on using them against their enemies.
HYDRA had Bucky Barnes train the Winter Soldiers; if they were to overpower him, they would be accepted into the program. During one session at the facility, Barnes fought and was quickly overpowered by Josef. As a doctor went to check on his vitals, the soldier responded by aggressively slammed his face and head onto the ground, killing him, prompting a guard to hit Josef with a baton. As a response, the five brainwashed Winter Soldiers then attacked their superiors, fighting the guards.
A terrified Vasily Karpov, having lost control of the Winter Soldiers, held Barnes as cover and demanded him to lead him to safety. While the soldiers continued to beat the guards to death, Barnes locked the soldiers inside the training session. Eventually, however, more guards were dispatched to the training room, and the Winter Soldiers were presumably subdued and knocked unconscious. HYDRA then decided to put them under suspended animation at the facility until their mind was controlled completely.
Many years later, Helmut Zemo would lose his family during the Battle of Sokovia, causing him to desire revenge against the Avengers. In 2016, the retired Sokovian general would use the Winter Soldier Book and other belongings he stole from Vasily Karpov to gain access to the facility. Despite what everyone thought, Zemo murdered the five Winter Soldiers, thus ending the Winter Soldier Program.