Pachulska-Wieczorek Katarzyna, Stefaniak Agnieszka K, Purzycka Katarzyna J
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznań, Poland.
Retrovirology. 2014 Jul 3;11:54. doi: 10.1186/1742-4690-11-54.
The nucleocapsid domain of Gag and mature nucleocapsid protein (NC) act as nucleic acid chaperones and facilitate folding of nucleic acids at critical steps of retroviral replication cycle. The basic N-terminus of HIV-1 NC protein was shown most important for the chaperone activity. The HIV-2 NC (NCp8) and HIV-1 NC (NCp7) proteins possess two highly conserved zinc fingers, flanked by basic residues. However, the NCp8 N-terminal domain is significantly shorter and contains less positively charged residues. This study characterizes previously unknown, nucleic acid chaperone activity of the HIV-2 NC protein.
We have comparatively investigated the in vitro nucleic acid chaperone properties of the HIV-2 and HIV-1 NC proteins. Using substrates derived from the HIV-1 and HIV-2 genomes, we determined the ability of both proteins to chaperone nucleic acid aggregation, annealing and strand exchange in duplex structures. Both NC proteins displayed comparable, high annealing activity of HIV-1 TAR DNA and its complementary nucleic acid. Interesting differences between the two NC proteins were discovered when longer HIV substrates, particularly those derived from the HIV-2 genome, were used in chaperone assays. In contrast to NCp7, NCp8 weakly facilitates annealing of HIV-2 TAR RNA to its complementary TAR (-) DNA. NCp8 is also unable to efficiently stimulate tRNALys3 annealing to its respective HIV-2 PBS motif. Using truncated NCp8 peptide, we demonstrated that despite the fact that the N-terminus of NCp8 differs from that of NCp7, this domain is essential for NCp8 activity.
Our data demonstrate that the HIV-2 NC protein displays reduced nucleic acid chaperone activity compared to that of HIV-1 NC. We found that NCp8 activity is limited by substrate length and stability to a greater degree than that of NCp7. This is especially interesting in light of the fact that the HIV-2 5'UTR is more structured than that of HIV-1. The reduced chaperone activity observed with NCp8 may influence the efficiency of reverse transcription and other key steps of the HIV-2 replication cycle.
Gag的核衣壳结构域和成熟核衣壳蛋白（NC）作为核酸伴侣，在逆转录病毒复制周期的关键步骤促进核酸折叠。HIV-1 NC蛋白的碱性N端对伴侣活性最为重要。HIV-2 NC（NCp8）和HIV-1 NC（NCp7）蛋白拥有两个高度保守的锌指结构，两侧为碱性残基。然而，NCp8的N端结构域明显较短，且带正电荷的残基较少。本研究对HIV-2 NC蛋白此前未知的核酸伴侣活性进行了表征。
我们比较研究了HIV-2和HIV-1 NC蛋白的体外核酸伴侣特性。使用来自HIV-1和HIV-2基因组的底物，我们测定了这两种蛋白在双链结构中伴侣核酸聚集、退火和链交换的能力。两种NC蛋白对HIV-1 TAR DNA及其互补核酸均表现出相当的高退火活性。当在伴侣分析中使用更长的HIV底物，特别是那些来自HIV-2基因组的底物时，发现了两种NC蛋白之间有趣的差异。与NCp7相反，NCp8对HIV-2 TAR RNA与其互补的TAR（-）DNA的退火促进作用较弱。NCp8也无法有效刺激tRNALys3与其各自的HIV-2 PBS基序退火。使用截短的NCp8肽，我们证明尽管NCp8的N端与NCp7不同，但该结构域对NCp8活性至关重要。
我们的数据表明，与HIV-1 NC相比，HIV-2 NC蛋白的核酸伴侣活性降低。我们发现，NCp8的活性比NCp7在更大程度上受底物长度和稳定性的限制。鉴于HIV-2 5'UTR比HIV-1的结构更复杂，这一点尤其有趣。观察到的NCp8伴侣活性降低可能会影响HIV-2复制周期的逆转录效率和其他关键步骤。
