scholarly journals Reverse Genetics with a Full-length Infectious cDNA Clone of Bovine Torovirus

2021 ◽  
Author(s):  
Makoto Ujike∗ ◽  
Yuka Etoh ◽  
Naoya Urushiyama ◽  
Fumihiro Taguchi ◽  
Hideki Asanuma ◽  
...  
Keyword(s):  
Viral Replication ◽  
Cdna Clone ◽  
Recombinant Virus ◽  
Reverse Genetics ◽  
Full Length ◽  
Egfp Expression ◽  
Egfp Gene ◽  
Virus Growth ◽  
Recombinant Viruses ◽  
Gene Retention

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified into the new family Tobaniviridae . While reverse genetics systems have been established for various CoVs, none exist for ToVs. Herein, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and HA-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated recombinant virus (rEGFP), wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. The rEGFP expressed EGFP in infected cells, but showed significantly lower viral growth compared to wtBToV. Moreover, the rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in non-structural proteins (NSPs) that emerged during passages exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a similar phenotype to these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. Importance ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae . Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, infection pathogenesis, and BToV vaccine development.

scholarly journals Reverse Genetics with a Full-length Infectious cDNA Clone of Bovine Torovirus

2020 ◽  
Author(s):  
Ujike Makoto ◽  
Etoh Yuka ◽  
Urushiyama Naoya ◽  
Taguchi Fumihiro ◽  
Enjuanes Luis ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cdna Clone ◽  
Recombinant Virus ◽  
Reverse Genetics ◽  
Infectious Cdna Clone ◽  
Full Length ◽  
Egfp Expression ◽  
Egfp Gene ◽  
Viral Growth ◽  
Recombinant Viruses

AbstractTorovirus (ToV) has recently been classified in the new family Tobaniviridae, although it belonged to the Coronavirus (CoV) family historically. Reverse genetics systems for many CoVs have been established, but none exist for ToVs. Here, we describe a reverse genetics system using a full-length infectious cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV containing genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the Hemagglutinin-esterase (HE) gene was manipulated, since cell-adapted wtBToV generally loses the full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and HA-tagged HEf or HEs genes were rescued; these showed no significant differences in cell growth, suggesting that HE is not essential for viral growth in cells. Then, recombinant virus in which HE was replaced by the Enhanced Green Fluorescent Protein (EGFP) gene expressed EGFP in infected cells, but showed significantly reduced viral growth compared to wtBToV. Moreover, the recombinant virus readily deleted the EGFP gene after one passage. Interestingly, one variant with mutations in non-structural proteins (NSPs) showed improved EGFP expression and viral growth during serial passages, although it eventually deleted the EGFP gene, suggesting that these mutations contributed to EGFP gene acceptance. These recombinant viruses provide new insights regarding BToV and its reverse genetics will help advance understanding of this neglected pathogen.ImportanceToVs are diarrhea-causing pathogens that have been detected in many species, including humans. BToV has spread worldwide, leading to economic losses. We developed the first reverse genetics system for Tobaniviridae using a BAC-based BToV. Using this system, we showed that recombinant BToVs with HEf and HEs showed no significant differences in cell growth. In contrast, clinical BToVs generally lose the HE gene after a few passages but some recombinant viruses retained the HE gene for up to 20 passages, suggesting some benefits of HE retention. The EGFP gene of the recombinant viruses was unstable and was rapidly deleted, likely via negative selection. Interestingly, one virus variant with mutations in NSPs was more stable, resulting in improved EGFP-expression and viral growth, suggesting that the mutations contributed to some acceptance of the exogenous EGFP gene without clear positive selection. The recombinant BToVs and reverse genetics developed here are powerful tools for understanding fundamental viral processes and their pathogenesis and for developing BToV vaccines.

scholarly journals User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome

Viruses ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 301
Author(s):  
Bingyu Yan ◽  
Xiaohui Zou ◽  
Xinglong Liu ◽  
Jiaming Zhao ◽  
Wenfeng Zhang ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Economic Losses ◽  
Virus Growth ◽  
Recombinant Viruses ◽  
Fowl Adenovirus ◽  
Viral Genes ◽  
Lmh Cells ◽  
Species Specific ◽  
The Right ◽  
User Friendly

A novel fowl adenovirus 4 (FAdV-4) has caused significant economic losses to the poultry industry in China since 2015. We established an easy-to-use reverse genetics system for modification of the whole right and partial left ends of the novel FAdV-4 genome, which worked through cell-free reactions of restriction digestion and Gibson assembly. Three recombinant viruses were constructed to test the assumption that species-specific viral genes of ORF4 and ORF19A might be responsible for the enhanced virulence: viral genes of ORF1, ORF1b and ORF2 were replaced with GFP to generate FAdV4-GFP, ORF4 was replaced with mCherry in FAdV4-GFP to generate FAdV4-GX4C, and ORF19A was deleted in FAdV4-GFP to generate FAdV4-CX19A. Deletion of ORF4 made FAdV4-GX4C form smaller plaques while ORF19A deletion made FAdV4-CX19A form larger ones on chicken LMH cells. Coding sequence (CDS) replacement with reporter mCherry demonstrated that ORF4 had a weak promoter. Survival analysis showed that FAdV4-CX19A-infected chicken embryos survived one more day than FAdV4-GFP- or FAdV4-GX4C-infected ones. The results illustrated that ORF4 and ORF19A were non-essential genes for FAdV-4 replication although deletion of either gene influenced virus growth. This work would help function study of genes on the right end of FAdV-4 genome and facilitate development of attenuated vaccines.

scholarly journals Reverse Genetic Generation of Recombinant Zaire Ebola Viruses Containing Disrupted IRF-3 Inhibitory Domains Results in Attenuated Virus Growth In Vitro and Higher Levels of IRF-3 Activation without Inhibiting Viral Transcription or Replication

2006 ◽  
Vol 80 (13) ◽  
pp. 6430-6440 ◽  
Author(s):  
Amy L. Hartman ◽  
Jason E. Dover ◽  
Jonathan S. Towner ◽  
Stuart T. Nichol
Keyword(s):  
Reverse Genetics ◽  
Ebola Virus ◽  
Viral Transcription ◽  
Type I ◽  
Virus Growth ◽  
Recombinant Viruses ◽  
Inhibitory Function ◽  
C Terminus ◽  
Inhibitory Domain

ABSTRACT The VP35 protein of Zaire Ebola virus is an essential component of the viral RNA polymerase complex and also functions to antagonize the cellular type I interferon (IFN) response by blocking activation of the transcription factor IRF-3. We previously mapped the IRF-3 inhibitory domain within the C terminus of VP35. In the present study, we show that mutations that disrupt the IRF-3 inhibitory function of VP35 do not disrupt viral transcription/replication, suggesting that the two functions of VP35 are separable. Second, using reverse genetics, we successfully recovered recombinant Ebola viruses containing mutations within the IRF-3 inhibitory domain. Importantly, we show that the recombinant viruses were attenuated for growth in cell culture and that they activated IRF-3 and IRF-3-inducible gene expression at levels higher than that for Ebola virus containing wild-type VP35. In the context of Ebola virus pathogenesis, VP35 may function to limit early IFN-β production and other antiviral signals generated from cells at the primary site of infection, thereby slowing down the host's ability to curb virus replication and induce adaptive immunity.

scholarly journals Reverse Genetics System Demonstrates that Rotavirus Nonstructural Protein NSP6 Is Not Essential for Viral Replication in Cell Culture

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Satoshi Komoto ◽  
Yuta Kanai ◽  
Saori Fukuda ◽  
Masanori Kugita ◽  
Takahiro Kawagishi ◽  
...  
Keyword(s):  
Cell Culture ◽  
Viral Replication ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Growth Curves ◽  
Single Step ◽  
Replication Cycle ◽  
Nonstructural Proteins ◽  
Virus Growth ◽  
Severe Diarrhea

ABSTRACT The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches.

scholarly journals A Reverse Genetics Platform That Spans the Zika Virus Family Tree

mBio ◽  
2017 ◽  
Vol 8 (2) ◽  
Author(s):  
Douglas G. Widman ◽  
Ellen Young ◽  
Boyd L. Yount ◽  
Kenneth S. Plante ◽  
Emily N. Gallichotte ◽  
...  
Keyword(s):  
Cell Culture ◽  
Latin America ◽  
Zika Virus ◽  
Recombinant Virus ◽  
Reverse Genetics ◽  
Family Tree ◽  
Immunodeficient Mice ◽  
Recombinant Viruses ◽  
Viral Emergence ◽  
The Caribbean

ABSTRACT Zika virus (ZIKV), a mosquito-borne flavivirus discovered in 1947, has only recently caused large outbreaks and emerged as a significant human pathogen. In 2015, ZIKV was detected in Brazil, and the resulting epidemic has spread throughout the Western Hemisphere. Severe complications from ZIKV infection include neurological disorders such as Guillain-Barré syndrome in adults and a variety of fetal abnormalities, including microcephaly, blindness, placental insufficiency, and fetal demise. There is an urgent need for tools and reagents to study the pathogenesis of epidemic ZIKV and for testing vaccines and antivirals. Using a reverse genetics platform, we generated six ZIKV infectious clones and derivative viruses representing diverse temporal and geographic origins. These include three versions of MR766, the prototype 1947 strain (with and without a glycosylation site in the envelope protein), and H/PF/2013, a 2013 human isolate from French Polynesia representative of the virus introduced to Brazil. In the course of synthesizing a clone of a circulating Brazilian strain, phylogenetic studies identified two distinct ZIKV clades in Brazil. We reconstructed viable clones of strains SPH2015 and BeH819015, representing ancestral members of each clade. We assessed recombinant virus replication, binding to monoclonal antibodies, and virulence in mice. This panel of molecular clones and recombinant virus isolates will enable targeted studies of viral determinants of pathogenesis, adaptation, and evolution, as well as the rational attenuation of contemporary outbreak strains to facilitate the design of vaccines and therapeutics. IMPORTANCE Viral emergence is a poorly understood process as evidenced by the sudden emergence of Zika virus in Latin America and the Caribbean. Malleable reagents that both predate and span an expanding epidemic are key to understanding the virologic determinants that regulate pathogenesis and transmission. We have generated representative cDNA molecular clones and recombinant viruses that span the known ZIKV family tree, including early Brazilian isolates. Recombinant viruses replicated efficiently in cell culture and were pathogenic in immunodeficient mice, providing a genetic platform for rational vaccine and therapeutic design. IMPORTANCE Viral emergence is a poorly understood process as evidenced by the sudden emergence of Zika virus in Latin America and the Caribbean. Malleable reagents that both predate and span an expanding epidemic are key to understanding the virologic determinants that regulate pathogenesis and transmission. We have generated representative cDNA molecular clones and recombinant viruses that span the known ZIKV family tree, including early Brazilian isolates. Recombinant viruses replicated efficiently in cell culture and were pathogenic in immunodeficient mice, providing a genetic platform for rational vaccine and therapeutic design.

scholarly journals A Point Mutation within the Replicase Gene Differentially Affects Coronavirus Genome versus Minigenome Replication

2005 ◽  
Vol 79 (24) ◽  
pp. 15016-15026 ◽  
Author(s):  
Carmen Galán ◽  
Luis Enjuanes ◽  
Fernando Almazán
Keyword(s):  
Amino Acid ◽  
Point Mutation ◽  
Cdna Clone ◽  
Reverse Genetics ◽  
Point Mutations ◽  
Full Length ◽  
Transmissible Gastroenteritis Virus ◽  
Replicase Gene ◽  
Full Length Cdna

ABSTRACT During the construction of the transmissible gastroenteritis virus (TGEV) full-length cDNA clone, a point mutation at position 637 that was present in the defective minigenome DI-C was maintained as a genetic marker. Sequence analysis of the recovered viruses showed a reversion at this position to the original virus sequence. The effect of point mutations at nucleotide 637 was analyzed by reverse genetics using a TGEV full-length cDNA clone and cDNAs from TGEV-derived minigenomes. The replacement of nucleotide 637 of TGEV genome by a T, as in the DI-C sequence, or an A severely affected virus recovery from the cDNA, yielding mutant viruses with low titers and small plaques compared to those of the wild type. In contrast, T or A at position 637 was required for minigenome rescue in trans by the helper virus. No relationship between these observations and RNA secondary-structure predictions was found, indicating that mutations at nucleotide 637 most likely had an effect at the protein level. Nucleotide 637 occupies the second codon position at amino acid 108 of the pp1a polyprotein. This position is predicted to map in the N-terminal polyprotein papain-like proteinase (PLP-1) cleavage site at the p9/p87 junction. Replacement of G-637 by A, which causes a drastic amino acid change (Gly to Asp) at position 108, affected PLP-1-mediated cleavage in vitro. A correlation was found between predicted cleaving and noncleaving mutations and efficient virus rescue from cDNA and minigenome amplification, respectively.

scholarly journals Construction of a Full-length Infectious Clone of Zika Virus Stably Expressing EGFP Marker in a Eukaryotic Expression System

2020 ◽  
Author(s):  
Jing Gao ◽  
Lingjuan Shi ◽  
Jiayi Chen ◽  
Weizhi Lu ◽  
Jingtai Cai ◽  
...  
Keyword(s):  
Zika Virus ◽  
Reverse Genetics ◽  
Infectious Clone ◽  
Expression System ◽  
Plaque Assay ◽  
Vero Cells ◽  
Full Length ◽  
Egfp Expression ◽  
Wild Type ◽  
Significant Difference

Abstract Background: Zika virus is among the most widely transmitted arboviruses in the world and closely associated with diseases, such as encephalitis, fetal microcephaly, and Guillain–Barré syndrome. The pathogenic mechanism of the virus has not been fully elucidated, and there are no vaccines or specific drugs targeting the virus. To address these issues, the application of reverse genetics is needed for viral reconstruction and reproduction.Methods: Polymerase chain reaction (PCR) was used to merge the full-length Zika virus genome, CMV promoter, intron, EGFP, hepatitis delta virus ribozyme, and SV40 terminator sequence for cloning into a pBAC11 vector through recombination to produce recombinant pBAC-ZIKA-EGFP. The ZIKA–EGFP was rescued by transfection of 293T cells with pBAC-ZIKA-EGFP, and at 7-days post-transfection, the supernatant (P0 generation) was passed through a 0.45-μm membrane and used to infect Vero cells (to produce the P1 generation). Fluorescence-based quantitative PCR, 50% tissue culture infectious dose, and plaque assays were used to measure differences in replication ability and pathogenicity relative to the rescue virus (ZIKA–WT), the sequence of which is consistent with that of the wild-type Zika virus. Additionally, caffeic acid phenethyl ester (CAPE), a nuclear factor kappaB (NF-kB) inhibitor, was used to examine its effect on viral replication.Results: The results showed that ZIKA–EGFP could effectively infect Vero cells, SH-SY5Y cells and C6/36 cells, and cause cytopathic effects on them. ZIKA–EGFP exhibited stable replication and EGFP expression during cell passage for at least six generations, with no significant difference in replication ability relative to the ZIKA–WT. Fluorescent cell foci were observed in the plaque assay while the ZIKA–EGFP was in the absence of phage plaque formation. The inhibition of NF-kB inhibitor on ZIKA-EGFP was observed by fluorescence microscopy, which was consistent with the results of fluorescence quantitative PCR.Conclusions: We constructed an infectious clone of the full-length genome of Zika virus which could replicate with stable EGFP expression in eukaryotic cells during passage. The infectious clone, remaining main characteristics of wild type ZIKA virus could be appied on the studies of reverse genetics, drug screening and gene function of ZIKA virus.

scholarly journals Enterovirus 71 VP1 Protein Regulates Viral Replication in SH-SY5Y Cells via the mTOR Autophagy Signaling Pathway

Viruses ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 11 ◽  
Author(s):  
Zi-Wei Liu ◽  
Zhi-Chao Zhuang ◽  
Rui Chen ◽  
Xiao-Rui Wang ◽  
Hai-Lu Zhang ◽  
...  
Keyword(s):  
Viral Replication ◽  
Virus Replication ◽  
Recombinant Virus ◽  
Enterovirus 71 ◽  
Nerve Cells ◽  
Full Length ◽  
Virulent Strains ◽  
Human Neurons ◽  
Virulence Attenuation ◽  
Full Length Gene

Background: Enterovirus 71 (EV71) is the main pathogen that causes severe hand, foot, and mouth disease with fatal neurological complications. However, its neurovirulence mechanism is still unclear. Candidate virulence sites were screened out at structural protein VP1, but the function of these candidate virulence sites remains unclear. Several studies have shown that autophagy is associated with viral replication. However, the relationship between VP1 and autophagy in human neurons has not been studied. Methods: A recombinant virus—SDLY107-VP1, obtained by replacing the VP1 full-length gene of the SDLY107 strain with the VP1 full-length gene of the attenuated strain SDJN2015-01—was constructed and tested for replication and virulence. We then tested the effect of the recombinant virus on autophagy in nerve cells. The effect of autophagy on virus replication was detected by western blot and plaque test. Finally, the changes of mTOR signaling molecules during EV71 infection and the effect of mTOR on virus replication at the RNA level were detected. Results: Viral recombination triggered virulence attenuation. The replication ability of recombinant virus SDLY107-VP1 was significantly weaker than that of the parent strain SDLY107. The SDLY107 strain could inhibit autophagic flux and led to accumulation of autophagosomes, while the SDLY107-VP1 strain could not cause autophagosome accumulation. The synthesis of EV71 RNA was inhibited by inhibiting mTOR. Conclusions: Replacement of VP1 weakened the replication ability of virulent strains and reduced the level of autophagy in nerve cells. This autophagy facilitates the replication of virulent strains in nerve cells. VP1 is an important neurovirulence determinant of EV71, which affects virus replication by regulating cell autophagy. mTOR is a key molecule in this type of autophagy.

scholarly journals Interdependence of Hemagglutinin Glycosylation and Neuraminidase as Regulators of Influenza Virus Growth: a Study by Reverse Genetics

2000 ◽  
Vol 74 (14) ◽  
pp. 6316-6323 ◽  
Author(s):  
Ralf Wagner ◽  
Thorsten Wolff ◽  
Astrid Herwig ◽  
Stephan Pleschka ◽  
Hans-Dieter Klenk
Keyword(s):  
Influenza Virus ◽  
Receptor Binding ◽  
Reverse Genetics ◽  
Mdck Cells ◽  
Influenza Virus Infection ◽  
Simian Virus 40 ◽  
Host Cells ◽  
Receptor Binding Site ◽  
Virus Growth ◽  
Recombinant Viruses

ABSTRACT The hemagglutinin (HA) of fowl plague virus A/FPV/Rostock/34 (H7N1) carries two N-linked oligosaccharides attached to Asn123 and Asn149 in close vicinity to the receptor-binding pocket. In previous studies in which HA mutants lacking either one (mutants G1 and G2) or both (mutant G1,2) glycosylation sites had been expressed from a simian virus 40 vector, we showed that these glycans regulate receptor binding affinity (M. Ohuchi, R. Ohuchi, A. Feldmann, and H. D. Klenk, J. Virol. 71:8377–8384, 1997). We have now investigated the effect of these mutations on virus growth using recombinant viruses generated by an RNA polymerase I-based reverse genetics system. Two reassortants of influenza virus strain A/WSN/33 were used as helper viruses to obtain two series of HA mutant viruses differing only in the neuraminidase (NA). Studies using N1 NA viruses revealed that loss of the oligosaccharide from Asn149 (mutant G2) or loss of both oligosaccharides (mutant G1,2) has a pronounced effect on virus growth in MDCK cells. Growth of virus lacking both oligosaccharides from infected cells was retarded, and virus yields in the medium were decreased about 20-fold. Likewise, there was a reduction in plaque size that was distinct with G1,2 and less pronounced with G2. These effects could be attributed to a highly impaired release of mutant progeny viruses from host cells. In contrast, with recombinant viruses containing N2 NA, these restrictions were much less apparent. N1 recombinants showed lower neuraminidase activity than N2 recombinants, indicating that N2 NA is able to partly overrule the high-affinity binding of mutant HA to the receptor. These results demonstrate that N-glycans flanking the receptor-binding site of the HA molecule are potent regulators of influenza virus growth, with the glycan at Asn149 being dominant and that at Asn123 being less effective. In addition, we show here that HA and NA activities need to be highly balanced in order to allow productive influenza virus infection.

scholarly journals The Kinetics of VP5 mRNA Expression Is Not Critical for Viral Replication in Cultured Cells

2000 ◽  
Vol 74 (6) ◽  
pp. 2770-2776 ◽  
Author(s):  
Pauline T. Lieu ◽  
Edward K. Wagner
Keyword(s):  
Viral Replication ◽  
Mrna Expression ◽  
Cultured Cells ◽  
Vero Cells ◽  
Lung Fibroblasts ◽  
Wild Type Virus ◽  
Wild Type ◽  
Virus Growth ◽  
Recombinant Viruses ◽  
Kinetics Of

ABSTRACT We generated recombinant viruses in which the kinetics of expression of the leaky-late VP5 mRNA was altered. We then analyzed the effect of such alterations on viral replication in cultured cells. The VP5 promoter and leader sequences from positions −36 to +20, containing the TATA box and an initiator element, were deleted and replaced with a strong early (dUTPase), an equal-strength leaky-late (VP16), or a strict-late (UL38) promoter. We found that recombinant viruses containing the dUTPase promoter inserted in the VP5 locus expressed VP5-encoding mRNA with early kinetics, while virus with the UL38 promoter inserted expressed such mRNA with strict-late kinetics. Further, in spite of differences in its functional architecture, the VP16 promoter fully substituted for the VP5 promoter. Western blot analysis demonstrated that the amounts of VP5 capsid protein produced by the recombinant viruses differed somewhat; however, on complementing C32 and noncomplementing Vero cells, such viruses replicated to titers equivalent to those of the rescued wild-type virus controls. Multistep virus growth in mouse embryo fibroblasts, rabbit skin cells, and Vero cells also demonstrated equivalent replication efficiencies for both recombinant and wild-type viruses. Further, recombinant viruses did not show any impairment in their ability to replicate on serum-starved or quiescent human lung fibroblasts. We conclude that the kinetics of the essential VP5 mRNA expression is not critical for viral replication in cultured cells.

Sign in / Sign up

Export Citation Format

Share Document