Replication differences of SARS-CoV-2 lineages may arise from unique RNA replication characteristics and nucleocapsid protein expression

Isadora Allonso Correa¹M Romario Matos de Souza¹Gustavo Peixoto Duarte da Silva¹Anna Beatriz Vianna Pimentel¹Pedro Telles Calil¹Marcela Sabino Cunha¹Diana Mariani²Rodrigo de Moraes Brindeiro²Sara Mesquita Costa³Maria Clara da Costa Simas³Victor Akira Ota⁴Elisa Cavalcante Pereira⁵Marilda Mendonça Siqueira⁵Paola Resende⁵Rafael Galliez⁶Debora Souza Faffe⁶Rosane Silva³Terezinha Marta Castiñeiras⁶Amilcar Tanuri⁷Luciana Jesus Da Costa^1*

• ¹Instituto de Microbiologia Paulo de Góes - Universidade Federal do Rio de Janeiro, Rio de Janeiro, Paraná, Brazil
• ²Departamento de Genética - Universidade Federal do Rio de Janeiro, Rio de Janeiro, Paraná, Brazil
• ³Instituto de Biofisica Carlos Chagas Filho Universidade Federal do Rio de Janeiro, Rio de Janeiro, Paraná, Brazil
• ⁴Nucleo de Enfrentamento e Estudos de Doenças Infecciosas Emergentes e Reeemergentes Universidade Federal do Rio de Janeiro, Nucleo de Enfrentamento de Doenças Infecciosas Emergentes e Reeemergentes Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
• ⁵Respiratory, Exantematic, Enterovirus and Viral Emergency Laboratory Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Rio de Janeiro, Brazil
• ⁶Nucleo de Enfrentamento de Doenças Infecciosas Emergentes e Reeemergentes Universidade Federal do Rio de Janeiro, Rio de Janeiro, Paraná, Brazil
• ⁷Departamento de Genética Universidade Federal do Rio de Janeiro, Rio de Janeiro, Paraná, Brazil

The COVID-19 pandemic was characterized by the sequential introduction and circulation of distinct SARS-CoV-2 variants, which presented differences in transmission capacity and pathogenicity. The relationship between these differences and the replicative capacity of these variants remains to be determined. Our research aimed to compare the biological traits of the SARS-CoV-2 lineages B.1.1.33, and variants Zeta (P.2), Gamma (P.1/P.1.*), Delta (B.1.617.2/AY.*), and Omicron (BA.*). We comprehensively demonstrated differences in virus entry, RNA replication, viral protein production, and virus infection progeny production amongst these variants. Importantly, these differences varied according to the cell type, with Omicron BA.1 exhibiting the lowest replication capacity in human pulmonary cells. Additionally, we demonstrated the occurrence of nucleocapsid proteoforms generated during infection and differences in size and number of sites of viral RNA replication for each virus, suggesting that factors beyond the initial stages of virus entry influence the efficiency of viral replication among different SARS-CoV-2 variants. Thus, our study underscores the significance of RNA replication and the role of nucleocapsid proteins in shaping the replicative characteristics of SARS-CoV-2 variants.