Lee Sook-Kyung, Nagashima Kunio, Hu Wei-Shau
HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, MD 21702, USA.
J Virol. 2005 Apr;79(7):4159-69. doi: 10.1128/JVI.79.7.4159-4169.2005.
The Gag polyprotein of murine leukemia virus (MLV) is processed into matrix (MA), p12, capsid (CA), and nucleocapsid (NC) proteins. p12 affects early events of virus replication and contains a PPPY motif important for virus release. To probe the functions of p12 in the early steps of MLV replication, we tested whether p12 can be replaced by spleen necrosis virus (SNV) p18, human immunodeficiency virus type 1 p6, or Rous sarcoma virus p2b. Analyses revealed that all chimeras generated virions at levels similar to that of MLV gag-pol; however, none of them could support MLV vector replication, and all of them exhibited severely reduced DNA synthesis upon virus infection. Because a previously reported SNV gag-MLV pol chimera, but not the MLV hybrid with SNV p18, can support replication of an MLV vector, we hypothesized that other Gag proteins act cooperatively with p12 during the early phase of virus replication. To test this hypothesis, we generated three more MLV-based chimeras containing SNV CA, p18-CA, or p18-CA-NC. We found that the MLV chimera containing SNV p18-CA or p18-CA-NC could support MLV vector replication, but the chimera containing SNV CA could not. Furthermore, viruses derived from the MLV chimera with SNV CA could synthesize viral DNA upon infection but were blocked at a post-reverse-transcription step and generated very little two long terminal repeat circle DNA, thereby producing a phenotype similar to that of the provirus formation-defective p12 mutants. Taken together, our data indicate that when p12/p18 or CA was from different viruses, despite abundant virus production and proper Gag processing, the resulting viruses were not infectious. However, when p12/p18 and CA were from the same virus, even though they were from SNV and not MLV, the resulting viruses were infectious. Therefore, these results suggest a cooperative effect of p12 and CA during the early events of MLV replication.
鼠白血病病毒（MLV）的Gag多聚蛋白被加工成基质（MA）、p12、衣壳（CA）和核衣壳（NC）蛋白。p12影响病毒复制的早期事件，并含有一个对病毒释放很重要的PPPY基序。为了探究p12在MLV复制早期步骤中的功能，我们测试了p12是否可以被脾坏死病毒（SNV）p18、人类免疫缺陷病毒1型p6或劳氏肉瘤病毒p2b替代。分析表明，所有嵌合体产生病毒粒子的水平与MLV gag-pol相似；然而，它们中没有一个能够支持MLV载体复制，并且在病毒感染后所有嵌合体的DNA合成均显著减少。由于先前报道的SNV gag-MLV pol嵌合体，但不是带有SNV p18的MLV杂种，能够支持MLV载体的复制，我们推测在病毒复制的早期阶段其他Gag蛋白与p12协同作用。为了验证这一假设，我们又构建了另外三种基于MLV的嵌合体，分别包含SNV CA、p18-CA或p18-CA-NC。我们发现含有SNV p18-CA或p18-CA-NC的MLV嵌合体能够支持MLV载体复制，但含有SNV CA的嵌合体则不能。此外，源自带有SNV CA的MLV嵌合体的病毒在感染后能够合成病毒DNA，但在逆转录后步骤受阻，并且产生的两个长末端重复序列环状DNA很少，从而产生了与前病毒形成缺陷型p12突变体相似的表型。综上所述，我们的数据表明，当p12/p18或CA来自不同病毒时，尽管产生了大量病毒且Gag加工正常，但产生的病毒没有传染性。然而，当p12/p18和CA来自同一病毒时，即使它们来自SNV而非MLV，产生的病毒仍具有传染性。因此，这些结果表明p12和CA在MLV复制早期事件中存在协同作用。
