FUNCTIONS OF THE PROTEINS OF HUMAN RESPIRATORY SYNCYTIAL VIRUS
人呼吸道合胞病毒蛋白质的功能
基本信息
- 批准号:6098950
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:DNA directed RNA polymerase RNA biosynthesis genetic transcription microorganism culture microorganism growth nucleocapsid phosphoproteins plasmids posttranslational modifications protein structure function replicase respiratory syncytial virus tissue /cell culture transfection virus RNA virus envelope virus genetics virus protein virus replication
项目摘要
Human respiratory syncytial virus (RSV), an
enveloped RNA- containing virus of the paramyxovirus family, is
the most important viral agent of pediatric respiratory tract disease.
Its genome is a single negative strand of RNA of 15,222
nucleotides that encodes ten major mRNAs. The viral genes are
transcribed in a sequential fashion in which transcript abundance
decreases with increasing distance of its gene from the promoter
(polar transcription). The purpose of this project is to identify the
functions of the viral proteins and to reconstruct events in the viral
growth cycle under conditions where they can be more readily
studied. Knowledge of the functions of the proteins is important for
the design of live- attenuated recombinant vaccine viruses. We
previously developed an intracellular transcription and replication
system for RSV based on components expressed from transfected
plasmid-borne cDNAs (see accompanying report). This involves
transfecting tissue culture cells with plasmids which individually
encode a helper-dependent minireplicon analog of negative-sense
genomic or positive-sense antigenomic RNA as well as whatever
mix of RSV proteins is desired. Three proteins, the nucleocapsid N
protein, phosphoprotein P and large polymerase subunit L protein,
constitute the RSV replicase whereas, unexpectedly, the
transcriptase requires in addition expression of the M2 mRNA. The
M2 mRNA contains two overlapping translational open reading
frames (ORFs), and the upstream one (M2-1) was shown to encode
a processivity factor. This is a completely novel finding for the
negative strand RNA viruses, and among RNA viruses such a factor
has only been described for human immunodeficiency virus 1,
namely the Tat protein. Additional experiments indicated that the
presence of M2-1 is not required for correct initiation, termination
or polyadenylation. However, in addition to its processivity activity,
the M2-1 protein was found to reduce the efficiency of
transcriptional termnation at the gene-end (GE) signal and thus
increases the amount of transcriptional readthrough. The effect of
M2-1 on RSV sequential transcription is being examined using
minigenomes which contain authentic groups of viral genes. Both
activities, namely processivity within genes and partial readthrough
of GE signals, has the effect of reducing the gradient of
transciptional polarity. Transcription and the first step of RNA
replication each involve the synthesis of positive-sense RNA off the
genomic template. It is generally assumed that the two processes
are interrelated and are regulated by the availability of N protein to
direct encapsidation of the antigenome. In the absence of sufficient
N protein, transcription would predominate. An alternative model is
that the M2-1 transcription factor might have a regulatory effect by
favoring transcription over replication. The reconstituted
transcription/replication system provided the first opportunity to
directly test these models. A series of tightly controlled experiments
showed that the levels of transcription and RNA replication are
insensitive to changes in the levels of N or of the other
nucleocapsid-associated proteins. The available data suggests that
these processes are independent rather than tightly interrelated.
Expression of the second ORF of the M2 mRNA (M2-2) inhibits
RNA replication and transcription, providing functional evidence
that this represents an eleventh RSV gene and is a novel negative
regulatory factor. We presently are working to detect this protein in
infected cells and to determine the mechanism by which it is
expressed from its overlapped ORF. The nonstructural protein NS1
also was found to be a potent inhibitor of RSV RNA synthesis. Like
the M2-2 protein, NS1 strongly inhibited transcription and both
steps of RNA replication. We found that it is possible to
reconstitute virion morphogenesis by coexpression of appropriate
envelope components with the above-mentioned transcription and
replication system. Morphogenesis was assayed by the passage of a
minireplicon to fresh cells. This showed that the matrix protein M,
attachment glycoprotein G and fusion glycoprotein F protein are
important for the formation of transmissible virus-like particles. The
small hydrophobic SH protein, both ORFs of the M2 mRNA, and
the nonstructural NS1 and NS2 proteins are completely dispensable
for particle formation. Interestingly, coexpression of the M2-1
transcription factor reduced the efficiency of virion packaging,
suggesting that a nucleocapsid which is engaged in fully processive
transcription might be refractory to packaging. Consistent with this,
the further coexpression of the M2-2 inhibitory factor increased the
efficiency of packaging and countered the effects of M2-1. Thus,
M2-2 might be a packaging factor.
人呼吸道合胞病毒(RSV)
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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PETER LEON COLLINS其他文献
PETER LEON COLLINS的其他文献
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{{ truncateString('PETER LEON COLLINS', 18)}}的其他基金
REPLICATION,VIRULENCE & IMMUNOGENICITY IN RECOMBINANT RESPIRATORY SYNCYTIAL V
复制、毒力
- 批准号:
6098927 - 财政年份:
- 资助金额:
-- - 项目类别:
STRUCTURAL ANALYSIS OF THE GENOME OF RESPIRATORY SYNCYTIAL VIRUS
呼吸道合胞病毒基因组的结构分析
- 批准号:
6288840 - 财政年份:
- 资助金额:
-- - 项目类别:
FUNCTIONS OF THE PROTEINS OF HUMAN RESPIRATORY SYNCYTIAL VIRUS
人呼吸道合胞病毒蛋白质的功能
- 批准号:
6288863 - 财政年份:
- 资助金额:
-- - 项目类别:
FUNCTIONS OF THE PROTEINS OF HUMAN RESPIRATORY SYNCYTIAL VIRUS
人呼吸道合胞病毒蛋白质的功能
- 批准号:
6431577 - 财政年份:
- 资助金额:
-- - 项目类别:
Paramyxoviruses as Vaccine Vectors Against Highly Pathogenic Viruses
副粘病毒作为高致病性病毒的疫苗载体
- 批准号:
7964502 - 财政年份:
- 资助金额:
-- - 项目类别:
Paramyxoviruses as Vaccine Vectors Against Highly Pathogenic Viruses
副粘病毒作为高致病性病毒的疫苗载体
- 批准号:
9566628 - 财政年份:
- 资助金额:
-- - 项目类别:
Laboratory Studies of Human Respiratory Syncytial Virus and Other Pneumoviruses
人类呼吸道合胞病毒和其他肺病毒的实验室研究
- 批准号:
8946258 - 财政年份:
- 资助金额:
-- - 项目类别:
Laboratory Studies of Human Respiratory Syncytial Virus and Other Pneumoviruses
人类呼吸道合胞病毒和其他肺病毒的实验室研究
- 批准号:
8745290 - 财政年份:
- 资助金额:
-- - 项目类别: