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标题: HBV 复制过程中的几个疑问，求解答。 [打印本页]

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作者: maxthon    时间: 2011-6-9 15:45     标题: HBV 复制过程中的几个疑问，求解答。



1. 松弛环状DNA进入细胞后要进入细胞核修复变成cccDNA。
请问，这个过程中是什么酶在起作用？这个酶是宿主的还是病毒本身的？
2.在细胞核里，cccDNA转录成mRNA使用的RNA聚合酶是宿主的还是病毒本身的？
3. mRNA逆转录生成单链-DNA的过程中使用的逆转录酶，是宿主的还是病毒本身的？
4. 从负链DNA生成双链DNA的过程中涉及到了DNA聚合酶，请问这个酶是宿主的还是病毒本身的？
5. 4中涉及到的DNA聚合酶与3中提及到的逆转录酶是同一种酶吗？蛋白结构是否相同？




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作者: lifevendor    时间: 2011-6-9 17:06

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1. 松弛环状DNA进入细胞后要进入细胞核修复变成cccDNA。 请问，这个过程中是什么酶在起作用？这个酶是宿主的还是病毒本身的？ “从分子生物学上来说， cccDNA的形成需要；a）除掉病毒的DNA聚合酶，该聚合酶是共价的连接在DNA的负链的5’端； b) 除掉5’端的一段连在正链的RNA oligomer低聚体(oligomer), RNAoligomer用于正链的DNA合成；c) 准确的去除掉负链多余的尾部 （short terminalredundancy）d)细胞内的复制机制完成正链的合成，注意在rcDNA中正链的初始长度是变化的e) ligation连接病毒的两端DNA（正链和负链）[4].” 理论上仅仅病毒核心颗粒进入宿主细胞核内，所以病毒核内那些东西都进入到细胞核，但是他们是否参与这个rcDNA-->cccDNA的过程不清楚，我猜是细胞核内的DNA 损害修复机制把rcDNA变成cccDNA，涉及到DNA repair, topoisomerase, 还有一些核小体组装的酶，这些酶应该都是宿主细胞核内的。‘‘Repair” of the incoming rcDNA: Completion of the plus strand of the rcDNA by the viral polymerase. Removal of the polymerase from the 5'-end of the minus strand DNA. Removal of a short RNA-primer used for the DNA- plus strand synthesis. Both processes are mediated by cellular enzymes [3]. 2.在细胞核里，cccDNA转录成mRNA使用的RNA聚合酶是宿主的还是病毒本身的？ 宿主的，这个可以肯定，cccDNA都变成微型核小体了 3. mRNA逆转录生成单链-DNA的过程中使用的逆转录酶，是宿主的还是病毒本身的？ 病毒的，这个在细胞核外完成，但在病毒核壳内，所以需要病毒表达rtPolymerase 4. 从负链DNA生成双链DNA的过程中涉及到了DNA聚合酶，请问这个酶是宿主的还是病毒本身的？ 这个是在细胞液中完成的，病毒的，还是刚才那个酶，我以前问过相同的问题。 5. 4中涉及到的DNA聚合酶与3中提及到的逆转录酶是同一种酶吗？蛋白结构是否相同？ 应该就是同一个酶，这个问题我以前也问过。这个聚合酶和pregenomicRNA被包在病毒核蛋白壳内然后完成接下来的所有的合成，理论上没有机会给其他的细胞液中的酶靠近。

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作者: lifevendor    时间: 2011-6-9 17:07

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http://www.hbvhbv.com/forum/thread-1022282-1-3.html也可以参考这个帖子

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作者: 抗战11    时间: 2011-6-9 19:05

路过，顶一下

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作者: interdetect    时间: 2011-6-10 08:20

lifevendor 发表于 2011-6-9 17:06
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3. mRNA逆转录生成单链-DNA的过程中使用的逆转录酶，是宿主的还是病毒本身的？
病毒的，这个在细胞核外完成，但在病毒核壳内，所以需要病毒表达rtPolymerase
4. 从负链DNA生成双链DNA的过程中涉及到了DNA聚合酶，请问这个酶是宿主的还是病毒本身的？
这个是在细胞液中完成的，病毒的，还是刚才那个酶，我以前问过相同的问题。

3、4这两个过程是在宿主细胞液里完成的，还是在病毒核壳里进行的呢？你能否把参考的文章全文发上来？

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作者: lifevendor    时间: 2011-6-10 09:04

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3，4两个过程描述的少，推荐看骆老的那本红宝书里面有详细的信息。
我贴两个英文的在这里：

”Encapsidation of pregenomic RNA
After translation of suffi cient amounts of HBc and
polymerase proteins, these proteins assemble together
with their mRNA, and cellular proteins, including the
heat shock protein Hsp90, the co chaperone p23, and
additional, as yet unknown, factors.143–147 Core particle
formation includes the encapsidation of a cellular protein
kinase, the identity of which is controversially discussed.
Encapsidation occurs only when the polymerase
interacts with a specifi c RNA sequence present at the 5′
end (base 1846 to base 1907) of the HBc mRNA.141 This
signal is termed ε (for encapsidation) and acts predominantly
in cis to the polymerase. ε is characterized by a
secondary structure consisting of a stem, a bulge, a loop
and a non-paired U (see also Fig. 10.9),265–267 but binding
of the polymerase alters the structure of ε268 and binding
to ε is required for activation of the polymerase.260 However,
fusions of ε to heterologous RNAs failed to cause
encapsidation. This observation initiated a search for
additional sequences required for packaging. Indeed, a
second region – termed ε II that is 900 nucleotides 3’ of ε
I – was found to be essential at least in avian hepadnaviruses.
269 In addition, this observation may explain why
only the pregenomic RNA is encapsidated, although the
signal sequence ε is present on the 3’ ends of all hepadnaviral
RNAs.



verse transcription to minus-strand DNA and plusstrand
DNA synthesis) is a tightly coupled event and
seems to occur exclusively within the core particles.
However, expression of an active DHBV polymerase
in a cell-free system showed that in these systems the
fi rst steps of DNA synthesis may occur in the absence
of core protein. Using this system, it could be shown
that minus-strand DNA synthesis starts in the bulge of
the ε-signal. In contrast to that of retroviruses, hepadnavirus
replication is initiated de novo, i.e. without a
nucleic acid primer. Instead, the –OH group of a tyrosine
in the amino-terminal domain of the polymerase
(Tyr 96 in DHBV,270 Tyr 63 in HBV41) is used for formation
of a phosphodiester linkage to the fi rst nucleotide.
Due to this mode of initiation, the polymerase
becomes and remains covalently linked to the DNA
minus-strand.



Elongation of DNA minus-strand
After synthesis of the fi rst four nucleotides within the
bulge (DHBV), the polymerase together with its covalently
bound nucleotides dissociates from its template
and re-anneals with a complementary sequence in the
DR1 close to the 3′ end, which was previously named
signal for reverse transcription (see Plate 10.1b [found
between p. 786–7] and Fig. 10.8). Experiments on the expression
of the HBV polymerase confi rmed this initiation
model for the human hepatitis B virus,271 although
the homology between the bulge and DR1 in all human
HBV genomes is restricted to three nucleotides. Because
there are many sequences complementary to the three
or four initial bases of the minus-DNA strand within
the hepadnavirus genome, an additional process must
be involved in correct transfer of the polymerase nucleotide
complex. Using mutagenesis analysis, a sequence
element designated phi located upstream of the 3’ DR1
sequence was identifi ed that is complementary to ε and
is important for effi cient viral replication. It was hypothesized
that this element brings the 3' DR1 sequence into
proximity with the three nucleotide primer synthesized
at the bulge of ε.272
Minus-strand DNA synthesis continues after priming
and translocation to the 3′ terminal DR1 of the
pregenomic RNA. From mapping the 3′ end of the minus-
DNA strand, it appears that this strand nearly coincides
with the 5′ end of the pregenomic RNA. Thus,
the 3′ end of the minus-DNA strand is specifi ed by ‘run
off’, when the polymerase reaches the end of its RNA
template, resulting in a terminal redundancy of the minus-
DNA strand of 8–10 nucleotides.



RNase H and priming of the DNA plus-strand
All reverse transcriptases are associated with an RNase
H activity, which cleaves the RNA of RNA–DNA hybrids
into oligoribonucleotides. Mutational inactivation
of the RNase H domain in the HBV polymerase results
in a block of DNA plus-strand strand synthesis.273 However,
the RNase H domain of HBV polymerase is not
able to cleave the last RNA nucleotides, bound to minus-
strand DNA. Thus, an 18-base-long capped RNA
fragment from the 5′ end of the pregenome is generated.
This fragment dissociates by unknown reasons from its
5′ terminal DR1 in the DNA minus-strand and is translocated
to DR2. Here, it functions as a primer for the
DNA plus-strand. The DNA begins with the last base
of DR2274,275 and continues towards the 5’ end on the
minus DNA strand. Here, the plus-DNA strand synthesis
crosses the discontinuity at the 3′ and 5′ ends of the
minus-DNA strand leading to a circular DNA genome.
Obviously, the specifi c primer translocation and the circularization
processes are complex. In addition to the
donor and acceptor sequences, three other cis-acting sequences,
named 3E, M and 5E, contribute to both processes.
By disrupting base-pairing between 3E and M3,
and between 5E and M5, evidence was obtained that
the ends of the minus-strand template are juxtaposedvia base-pairing to facilitate the two template switches
during plus-strand DNA synthesis.276 However, a small
terminal redundancy (5’r and 3’r) on the ends of the minus-
strand DNA has also been shown to be important,
but not suffi cient, for circularization.277,278 “ （from Viral Hepatitis 3rd Version)

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作者: lifevendor    时间: 2011-6-10 09:09

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[attach]222288[/attach]

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作者: StephenW    时间: 2011-6-10 11:49



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请观察第11步，12步，13步在Lifevendor的图。
[attach]222300[/attach]
(11) Translation of the pregenomic RNA (pgRNA) to the core
protein and the viral polymerase. The regulatory X-protein
and the three envelope proteins are translated from the
subgenomic RNAs.
(12) Complex formation of the pgRNA (via its epsilon stemloop
structure) with the core protein and the polymerase
and self-assembly of an RNA-containing nucleocapsid.
(13) Reverse transcription of the pgRNA followed by plusstrand
DNA-synthesis within the nucleocapsid. Maturation
of the RNA-containing nucleocapsids to DNA-containing
nucleocapsids within the cytoplasm.

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作者: interdetect    时间: 2011-9-5 15:48

StephenW 发表于 2011-6-10 11:49
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请观察第11步，12步，13步在Lifevendor的图。

where did you get this picture?
Can you help me ?
Thank you.

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作者: StephenW    时间: 2011-9-5 16:45



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Can have a look at these:
http://www.jhep-elsevier.com/article/PIIS0168827809007387/fulltext
http://www.docin.com/p-83055441.html

Most of my images are from Google Image search using keywords such as "hbv", "lamivudine", etc.

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