RNA replication by a respiratory syncytial virus RNA analog does not obey the rule of six and retains a nonviral trinucleotide extension at the leader end

被引:69
作者
Samal, SK
Collins, PL
机构
[1] NIAID,NIH,INFECT DIS LAB,BETHESDA,MD 20892
[2] UNIV MARYLAND,REG COLL VET MED,COLLEGE PK,MD 20742
关键词
D O I
10.1128/JVI.70.8.5075-5082.1996
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Genome analogs (''minigenomes'') of Sendai and measles viruses replicate efficiently only if their nucleotide length is an even multiple of six, a requirement called the rule of six (P. Calain and L. Roux, J. Virol. 67:4822-4830, 1993; M. S. Sidhu, J. Chan, K. Kaelin, P. Spielhofer, F. Radecke, H. Schneider, M. Masurekar, P. C. Dowling, M. A. Billeter, and S. A. Udem, Virology 208:800-807, 1995). The existence of a comparable requirement was tested for respiratory syncytial virus (RSV), which also is a member of family Paramyxoviridae and whose natural genome length also is a multiple of six. An internally truncated analog of RSV positive-sense replicative intermediate RNA (antigenome) bearing the chloramphenicol acetyltransferase gene as a reporter was synthesized from cDNA in vitro. This RNA was transfected into cells which were infected with RSV as a helper. Miniantigenomic RNA was indistinguishable from previously studied negative-sense minigenome RNA in its ability to participate in transcription, RNA replication, and incorporation into transmissible particles. Sixteen miniantigenomes which were of slightly different lengths and which in aggregate represented multiples of a wide range of integers including 1 to 15 were constructed. During transfection and two serial passages, the various miniantigenomes were essentially indistinguishable with regard to the efficiency of transcription, RNA replication, and packaging into transmissible particles. Progeny minigenomes of six different mutants were recovered postpassage, copied into cDNA, cloned, and sequenced completely. The length of each of these RNAs was found to have remained unchanged during replication and passage. Thus, RSV transcription and replication appear to lack the requirement that the template length be an even multiple of an integer such as six, which for Sendai and measles viruses is obligatory for nucleocapsid function. Each of the in vitro-synthesized miniantigenomes used in transfection contained a nonviral extension of three nucleotides, GGG,,on the 5' (leader) end contributed by the T7 promoter. The termini of the recovered minigenomes were examined for five mutants by RNA circularization followed by cDNA synthesis, amplification, cloning, and sequencing. Unexpectedly, each recovered minigenome contained the complement of this nonviral extension on the 3' (leader) end, showing that it had been faithfully copied and maintained during RNA replication and passage. The nonviral trinucleotide did not appear to affect the activity of the template.
引用
收藏
页码:5075 / 5082
页数:8
相关论文
共 40 条
[1]   MOLECULAR STUDIES OF BROME MOSAIC-VIRUS USING INFECTIOUS TRANSCRIPTS FROM CLONED CDNA [J].
AHLQUIST, P ;
FRENCH, R ;
BUJARSKI, JJ .
ADVANCES IN VIRUS RESEARCH, 1987, 32 :215-242
[2]  
BAUSCH JN, 1984, J BIOL CHEM, V258, P1978
[3]   FUNCTIONAL-CHARACTERIZATION OF THE GENOMIC AND ANTIGENOMIC PROMOTERS OF SENDAI VIRUS [J].
CALAIN, P ;
ROUX, L .
VIROLOGY, 1995, 212 (01) :163-173
[4]   THE RULE OF 6, A BASIC FEATURE FOR EFFICIENT REPLICATION OF SENDAI VIRUS DEFECTIVE INTERFERING RNA [J].
CALAIN, P ;
ROUX, L .
JOURNAL OF VIROLOGY, 1993, 67 (08) :4822-4830
[5]   ONE-HOUR DOWNWARD ALKALINE CAPILLARY TRANSFER FOR BLOTTING OF DNA AND RNA [J].
CHOMCZYNSKI, P .
ANALYTICAL BIOCHEMISTRY, 1992, 201 (01) :134-139
[6]  
COLLINS PG, UNPUB
[7]   Transcription elongation factor of respiratory syncytial virus, a nonsegmented negative-strand RNA virus [J].
Collins, PL ;
Hill, MG ;
Cristina, J ;
Grosfeld, H .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1996, 93 (01) :81-85
[8]   RESCUE OF A 7502-NUCLEOTIDE (49.3-PERCENT OF FULL-LENGTH) SYNTHETIC ANALOG OF RESPIRATORY SYNCYTIAL VIRUS GENOMIC RNA [J].
COLLINS, PL ;
MINK, MA ;
HILL, MG ;
CAMARGO, E ;
GROSFELD, H ;
STEC, DS .
VIROLOGY, 1993, 195 (01) :252-256
[9]   RESCUE OF SYNTHETIC ANALOGS OF RESPIRATORY SYNCYTIAL VIRUS GENOMIC RNA AND EFFECT OF TRUNCATIONS AND MUTATIONS ON THE EXPRESSION OF A FOREIGN REPORTER GENE [J].
COLLINS, PL ;
MINK, MA ;
STEC, DS .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1991, 88 (21) :9663-9667
[10]   PRODUCTION OF INFECTIOUS HUMAN RESPIRATORY SYNCYTIAL VIRUS FROM CLONED CDNA CONFIRMS AN ESSENTIAL ROLE FOR THE TRANSCRIPTION ELONGATION-FACTOR FROM THE 5'-PROXIMAL OPEN READING FRAME OF THE M2 MESSENGER-RNA IN GENE-EXPRESSION AND PROVIDES A CAPABILITY FOR VACCINE DEVELOPMENT [J].
COLLINS, PL ;
HILL, MG ;
CAMARGO, E ;
GROSFELD, H ;
CHANOCK, RM ;
MURPHY, BR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1995, 92 (25) :11563-11567