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1.
We have previously described a new family of mutant adenoviruses carrying different combinations of attB/attP sequences from bacteriophage PhiC31 flanking the Ad5 packaging domain. These novel helper viruses have a significantly delayed viral life cycle and a severe packaging impairment, regardless of the presence of PhiC31 recombinase. Their infectious viral titers are significantly lower (100-1000 fold) than those of control adenovirus at 36 hours post-infection, but allow for efficient packaging of helper-dependent adenovirus. In the present work, we have analyzed which steps of the adenovirus life cycle are altered in attB-helper adenoviruses and investigated whether these viruses can provide the necessary viral proteins in trans. The entry of attB-adenoviral genomes into the cell nucleus early at early timepoints post-infection was not impaired and viral protein expression levels were found to be similar to those of control adenovirus. However, electron microscopy and capsid protein composition analyses revealed that attB-adenoviruses remain at an intermediate state of maturation 36 hours post-infection in comparison to control adenovirus which were fully mature and infective at this time point. Therefore, an additional 20-24 hours were found to be required for the appearance of mature attB-adenovirus. Interestingly, attB-adenovirus assembly and infectivity was restored by inserting a second packaging signal close to the right-end ITR, thus discarding the possibility that the attB-adenovirus genome was retained in a nuclear compartment deleterious for virus assembly. The present study may have substantive implications for helper-dependent adenovirus technology since helper attB-adenovirus allows for preferential packaging of helper-dependent adenovirus genomes.  相似文献   

2.
Human immunodeficiency virus type 2 (HIV-2) has been reported to have a distinct RNA packaging mechanism, referred to as cis packaging, in which Gag proteins package the RNA from which they were translated. We examined the progeny generated from dually infected cell lines that contain two HIV-2 proviruses, one with a wild-type gag/gag-pol and the other with a mutant gag that cannot express functional Gag/Gag-Pol. Viral titers and RNA analyses revealed that mutant viral RNAs can be packaged at efficiencies comparable to that of viral RNA from which wild-type Gag/Gag-Pol is translated. These results do not support the cis-packaging hypothesis but instead indicate that trans packaging is the major mechanism of HIV-2 RNA packaging. To further characterize the mechanisms of HIV-2 RNA packaging, we visualized HIV-2 RNA in individual particles by using fluorescent protein-tagged RNA-binding proteins that specifically recognize stem-loop motifs in the viral genomes, an assay termed single virion analysis. These studies revealed that >90% of the HIV-2 particles contained viral RNAs and that RNAs derived from different viruses were copackaged frequently. Furthermore, the frequencies of heterozygous particles in the viral population could be altered by changing a 6-nucleotide palindromic sequence at the 5'-untranslated region of the HIV-2 genome. This finding indicates that selection of copackaging RNA partners occurs prior to encapsidation and that HIV-2 Gag proteins primarily package one dimeric RNA rather than two monomeric RNAs. Additionally, single virion analyses demonstrated a similar RNA distribution in viral particles regardless of whether both viruses had a functional gag or one of the viruses had a nonfunctional gag, providing further support for the trans-packaging hypothesis. Together, these results revealed mechanisms of HIV-2 RNA packaging that are, contrary to previous studies, in many respects surprisingly similar to those of HIV-1.  相似文献   

3.
Helper-dependent (HD) adenovirus vectors devoid of all viral coding sequences have a large cloning capacity and provide long-term transgene expression in vivo with negligible toxicity, making them attractive vectors for gene therapy. Currently, the most efficient means of producing HD vectors involves coinfecting 293 cells expressing Cre with the HD vector and a helper virus bearing a packaging signal flanked by loxP sites. Cre-mediated packaging signal excision renders the helper virus genome unpackageable but still able to replicate and provide helper functions for HD vector propagation. Typically, helper virus contamination is < or =1% pre- and < or =0.1% postpurification by CsCl banding. While these contamination levels are low, further reduction is desirable. However, this objective has not been realized since the Cre/loxP system was first developed. This lack of progress is due, at least in part, to our lack of understanding of the origins of the contaminating helper virus, thus rendering its reduction or elimination difficult to achieve. This study was designed to investigate the possible sources of contaminating helper virus persisting during HD vector amplification. The results revealed that Cre is limiting in helper virus-infected Cre-expressing 293 cells, thereby permitting helper viruses to escape packaging signal excision and propagate. The results of this study should provide a foundation for developing rational strategies to further reduce or possibly eliminate the contaminating helper virus.  相似文献   

4.
Ma HC  Hearing P 《Journal of virology》2011,85(15):7849-7855
The packaging of the adenovirus (Ad) genome into a capsid displays serotype specificity. This specificity has been attributed to viral packaging proteins, the IVa2 protein and the L1-52/55K protein. We previously found that the Ad17 L1-52/55K protein was not able to complement the growth of an Ad5 L1-52/55K mutant virus, whereas two other Ad17 packaging proteins, IVa2 and L4-22K, could complement the growth of Ad5 viruses with mutations in the respective genes. In this report, we investigated why the Ad17 L1-52/55K protein was not able to complement the Ad5 L1-52/55K mutant virus. We demonstrate that the Ad17 L1-52/55K protein binds to the Ad5 IVa2 protein in vitro and the Ad5 packaging domain in vivo, activities previously associated with packaging function. The Ad17 L1-52/55K protein also associates with empty Ad5 capsids. Interestingly, we find that the Ad17 L1-52/55K protein is able to complement the growth of an Ad5 L1-52/55K mutant virus in conjunction with the Ad17 structural protein IIIa. The same result was found with the L1-52/55K and IIIa proteins of several other Ad serotypes, including Ad3 and Ad4. The Ad17 IIIa protein associates with empty Ad5 capsids. Consistent with the complementation results, we find that the IIIa protein interacts with the L1-52/55K protein in vitro and associates with the viral packaging domain in vivo. These results underscore the complex nature of virus assembly and genome encapsidation and provide a new model for how the viral genome may tether to the empty capsid during the encapsidation process.  相似文献   

5.
Novel retroviral protein expression constructs were designed to retain minimal retroviral sequences and to express dominant selectable markers by reinitiation of translation after expression of the viral genes. HT1080 cells were selected as producer cells for their ability to release high-titer viruses that are resistant to inactivation by human serum. Two HT1080-based packaging cell lines which produce Moloney murine leukemia virus cores with envelope glycoproteins of either amphotropic murine leukemia virus (FLYA13 line) or cat endogenous virus RD114 (FLYRD18 line) are described. Direct comparison with previous retroviral packaging systems indicated that 100-fold-higher titers of helper-free recombinant viruses were released by the FLYA13 and FLYRD18 lines.  相似文献   

6.
Construction of adenovirus vectors through Cre-lox recombination.   总被引:19,自引:0,他引:19       下载免费PDF全文
Two barriers prevent adenovirus-based vectors from having wide application. One is the difficulty of making new adenoviruses, and the second is the strong immunological reaction to viral proteins. Here we describe uses of Cre-lox recombination to overcome these problems. First, we demonstrate a simple method for constructing E1-substituted adenoviruses. Second, we demonstrate a method to construct adenovirus vectors carrying recombinant genes in place of all of the viral genes, so-called gutless adenovirus vectors. The pivotal feature in each method is the use of a negatively selected adenovirus named psi5. We engineered a cis-acting selection into psi5 by flanking its packaging site with loxP sites. When psi5 was grown in cells making a high level of Cre recombinase, the packaging site was deleted by recombination and the yield of psi5 was reduced to 5% of the wild-type level. To make a new E1-substituted virus, we used psi5 as a donor virus and recombined it with a shuttle vector via a loxP site. The resulting recombinant virus has a single loxP site next to the packaging site and therefore outgrows psi5 in the presence of Cre recombinase. To make a gutless virus, we used psi5 as a helper virus. The only viral sequences included in the gutless vector are those needed in cis for its replication and packaging. We found that a loxP site next to the packaging site of the gutless virus was necessary to neutralize homologous recombination between psi5 and the gutless viruses within their packaging domains.  相似文献   

7.
Yang K  Wills EG  Baines JD 《Journal of virology》2011,85(22):11972-11980
Herpesvirus genomic DNA is cleaved from concatemers that accumulate in infected cell nuclei. Genomic DNA is inserted into preassembled capsids through a unique portal vertex. Extensive analyses of viral mutants have indicated that intact capsids, the portal vertex, and all components of a tripartite terminase enzyme are required to both cleave and package viral DNA, suggesting that DNA cleavage and packaging are inextricably linked. Because the processes have not been functionally separable, it has been difficult to parse the roles of individual proteins in the DNA cleavage/packaging reaction. In the present study, a virus bearing the deletion of codons 400 to 420 of U(L)15, encoding a terminase component, was analyzed. This virus, designated vJB27, failed to replicate on noncomplementing cells but cleaved concatemeric DNA to ca. 35 to 98% of wild-type levels. No DNA cleavage was detected in cells infected with a U(L)15-null virus or a virus lacking U(L)15 codons 383 to 385, comprising a motif proposed to couple ATP hydrolysis to DNA translocation. The amount of vJB27 DNA protected from DNase I digestion was reduced compared to the wild-type virus by 6.5- to 200-fold, depending on the DNA fragment analyzed, thus indicating a profound defect in DNA packaging. Capsids containing viral DNA were not detected in vJB27-infected cells, as determined by electron microscopy. These data suggest that pU(L)15 plays an essential role in DNA translocation into the capsid and indicate that this function is separable from its role in DNA cleavage.  相似文献   

8.
Given their generous transgene capacity and inherent neurotropism, herpes simplex virus (HSV-1)-based viral vectors are promising tools for gene delivery to the central nervous system. Despite their widespread pre-clinical use, vector toxicity remains a concern with regard to the use of herpes vectors in humans. One potential source of toxicity stems from the tegument-associated virion host shutoff protein (vhs), which induces translational arrest in the host cell through non-specific mRNAse activity. In the current study we utilized a series of HSV-1 viruses containing a deletion in the U(L)41 open reading frame to investigate: (1) the requirement of intact vhs function in amplicon packaging and (2) whether vhs influences the post-transduction survival of dissociated cortical neurons. Our results demonstrate that while amplicon yield was reduced an order of magnitude, U(L)41 deletion was associated with reduced vector toxicity. Furthermore, partial reconstitution of vhs function using mRNAse-inactive point mutants improved amplicon titers without imparting the toxicity observed with wild-type controls. These findings offer a novel approach to improving the titer and toxicity profiles of HSV-based viral vectors.  相似文献   

9.
A final step in the influenza virus replication cycle is the assembly of the viral structural proteins and the packaging of the eight segments of viral RNA (vRNA) into a fully infectious virion. The process by which the RNA genome is packaged efficiently remains poorly understood. In an approach to analyze how vRNA is packaged, we rescued a seven-segmented virus lacking the hemagglutinin (HA) vRNA (deltaHA virus). This virus could be passaged in cells constitutively expressing HA protein, but it was attenuated in comparison to wild-type A/WSN/33 virus. Supplementing the deltaHA virus with an artificial segment containing green fluorescent protein (GFP) or red fluorescent protein (RFP) with HA packaging regions (45 3' and 80 5' nucleotides) partially restored the growth of this virus to wild-type levels. The absence of the HA vRNA in the deltaHA virus resulted in a 40 to 60% reduction in the packaging of the PA, NP, NA, M, and NS vRNAs, as measured by quantitative PCR (qPCR), and the packaging of these vRNAs was partially restored in the presence of GFP/RFP packaging constructs. To further define nucleotides of the HA coding sequence which are important for vRNA packaging, synonymous mutations were introduced into the full-length HA cDNA of influenza A/WSN/33 and A/Puerto Rico/8/34 viruses, and mutant viruses were rescued. qPCR analysis of vRNAs packaged in these mutant viruses identified a key region of the open reading frame (nucleotides 1659 to 1671) that is critical for the efficient packaging of an influenza virus H1 HA segment.  相似文献   

10.
The design of drugs for treatment of virus infections and the exploitation of viruses as drugs for treatment of diseases could be made more successful by understanding the molecular mechanisms of virus-specific events. The process of assembly, and more specifically packaging of the genome into a capsid, is an obligatory step leading to future infections. To enhance our understanding of the molecular mechanism of packaging, it is necessary to characterize the viral components necessary for the event. In the case of adenovirus, sequences between nucleotides 200 and 400 at the left end of the genome are essential for packaging. This region contains a series of redundant bipartite sequences, termed A repeats, that function in packaging. Synthetic packaging sequences made of multimers of a single A repeat substitute for the authentic adenovirus packaging domain. A repeats are binding sites for the CCAAT displacement protein and the viral protein IVa2. Several lines of evidence implicate these proteins in the packaging process. It was not known, however, whether other cis-acting elements play a role in the packaging process as well. We utilized an in vivo approach to address the role of the inverted terminal repeats and the covalently linked terminal proteins in packaging of the adenovirus genome. Our results show that these elements are not necessary for efficient packaging of the viral genome. A significant implication of these results applicable to gene therapy vector design is that the linkage of the adenovirus packaging domain to heterologous DNA sequences should suffice for targeting to the viral capsid.  相似文献   

11.
S Oertle  P F Spahr 《Journal of virology》1990,64(12):5757-5763
Rous sarcoma virus nucleocapsid protein (NC) has been shown by site-directed mutagenesis to be involved in viral RNA packaging and in the subsequent maturation of genomic RNA in the progeny viral particles. To investigate whether NC exerts these activities as a free protein or as a domain of the polyprotein precursor Pr76gag, we have constructed several mutants unable to process Pr76gag and analyzed their properties in a transient-transfection assay of chicken embryo fibroblasts, the natural host of Rous sarcoma virus. A point mutation in the protease (PR) active site completely prevents Pr76gag processing. The full-length Pr76gag polyprotein is still able to package viral RNA, but cannot mature it. A shorter gag precursor polyprotein lacking the C-terminal PR domain, but retaining that of the NC protein, is however, unable even to package viral RNA. This indicates that the NC protein can participate in packaging viral RNA only as part of a full-length Pr76gag and that the PR domain is, indirectly or directly, also involved in RNA packaging. These results also demonstrate that processing of Pr76gag is necessary for viral RNA dimerization.  相似文献   

12.
The molecular mechanism for packaging of the adenovirus (Ad) genome into the capsid is likely similar to that of DNA bacteriophages and herpesviruses-the insertion of viral DNA through a portal structure into a preformed prohead driven by an ATP-hydrolyzing molecular machine. It is speculated that the IVa2 protein of adenovirus is the ATPase providing the power stroke of the packaging machinery. Purified IVa2 binds ATP in vitro and, along with a second Ad protein, the L4 22-kilodalton protein (L4-22K), binds specifically to sequences in the Ad genome that are essential for packaging. The efficiency of binding of these proteins in vitro was correlated with the efficiency of packaging in vivo. By utilizing a virus unable to express IVa2, pm8002, it was reported that IVa2 plays a role in assembly of the empty virion. We wanted to address the question of whether the ATP binding, and hence the putative ATPase activity, of IVa2 was required for its role in virus assembly. Our results show that ATPase activity was not required for the assembly of empty virus particles. In addition, we present evidence that particles were assembled in the absence of IVa2 by using two viruses null for IVa2-a deletion mutant virus, ΔIVa2, and the previously described mutant virus, pm8002. Empty virus particles produced by these IVa2 mutant viruses did not contain detectable viral DNA. We conclude that the major role of IVa2 is in viral DNA packaging. A characterization of the empty particles obtained from the IVa2 mutant viruses compared to wild-type empty particles is presented.  相似文献   

13.
Selectivity and polarity of adenovirus type 5 DNA packaging are believed to be directed by an interaction of putative packaging factors with the cis-acting adenovirus packaging domain located within the genomic left end (nucleotides 194 to 380). In previous studies, this packaging domain was mutationally dissected into at least seven functional elements called A repeats. These elements, albeit redundant in function, exhibit differences in the ability to support viral packaging, with elements I, II, V, and VI as the most critical repeats. Viral packaging was shown to be sensitive to spatial changes between individual A repeats. To study the importance of spatial constraints in more detail, we performed site-directed mutagenesis of the 21-bp linker regions separating A repeats I and II, as well as A repeats V and VI. The results of our mutational analysis reveal previously unrecognized sequences that are critical for DNA encapsidation in vivo. On the basis of these results, we present a more complex consensus motif for the adenovirus packaging elements which is bipartite in structure. DNA encapsidation is compromised by changes in spacing between the two conserved parts of the consensus motif, rather than between different A repeats. Genetic evidence implicating packaging elements as independent units in viral DNA packaging is derived from the selection of revertants from a packaging-deficient adenovirus: multimerization of packaging repeats is sufficient for the evolution of packaging-competent viruses. Finally, we identify minimally sized segments of the adenovirus packaging domain that can confer viability and packaging activity to viruses carrying gross truncations within their left-end sequences. Coinfection experiments using the revertant as well as the minimal-packaging-domain mutant viruses strengthen existing arguments for the involvement of limiting, trans-acting components in viral DNA packaging.  相似文献   

14.
15.
16.
Adenovirus type 5 (Ad5) DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent on the cis-acting packaging domain located between nucleotides 230 and 380. Seven AT-rich repeats that direct packaging have been identified within this domain. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans-acting factor(s) that plays a role in packaging. Both cellular and viral proteins that interact with adenovirus packaging elements in vitro have been identified. In this study, we characterized a group of recombinant viruses that carry site-specific point mutations within a minimal packaging domain. The mutants were analyzed for growth properties in vivo and for the ability to bind cellular and viral proteins in vitro. Our results are consistent with a requirement of the viral IVa2 protein for DNA packaging via a direct interaction with packaging sequences. Our results also indicate that higher-order IVa2-containing complexes that form on adjacent packaging repeats in vitro are the complexes required for the packaging activity of these sites in vivo. Chromatin immunoprecipitation was used to study proteins that bind directly to the packaging sequences. These results demonstrate site-specific interaction of the viral IVa2 and L1 52/55K proteins with the Ad5 packaging domain in vivo. These results confirm and extend those previously reported and provide a framework on which to model the adenovirus assembly process.  相似文献   

17.
Reovirus attachment protein σ1 is an elongated trimer with head-and-tail morphology that engages cell-surface carbohydrate and junctional adhesion molecule A (JAM-A). The σ1 protein is comprised of three domains partitioned by two flexible linkers termed interdomain regions (IDRs). To determine the importance of σ1 length and flexibility at different stages of reovirus infection, we generated viruses with mutant σ1 molecules of altered length and flexibility and tested these viruses for the capacity to bind the cell surface, internalize, uncoat, induce protein synthesis, assemble, and replicate. We reduced the length of the α-helical σ1 tail to engineer mutants L1 and L2 and deleted midpoint and head-proximal σ1 IDRs to generate ΔIDR1 and ΔIDR2 mutant viruses, respectively. Decreasing length or flexibility of σ1 resulted in delayed reovirus infection and reduced viral titers. L1, L2, and ΔIDR1 viruses but not ΔIDR2 virus displayed reduced cell attachment, but altering σ1 length or flexibility did not diminish the efficiency of virion internalization. Replication of ΔIDR2 virus was hindered at a postdisassembly step. Differences between wild-type and σ1 mutant viruses were not attributable to alterations in σ1 folding, as determined by experiments assessing engagement of cell-surface carbohydrate and JAM-A by the length and IDR mutant viruses. However, ΔIDR1 virus harbored substantially less σ1 on the outer capsid. Taken together, these data suggest that σ1 length is required for reovirus binding to cells. In contrast, IDR1 is required for stable σ1 encapsidation, and IDR2 is required for a postuncoating replication step. Thus, the structural architecture of σ1 is required for efficient reovirus infection of host cells.  相似文献   

18.
Previous analyses have demonstrated that adenovirus DNA is packaged into virions in vivo in a polar, left-to-right fashion. The packaging of viral DNA is dependent on cis-acting elements at the left end of the genome. In this report, we describe a genetic analysis of the sequences that are required for efficient packaging of adenovirus type 5 (Ad5) DNA. Our results demonstrate that the Ad5 packaging domain (nucleotides 194 to 358) is composed of at least five distinct elements that are functionally redundant. An AT-rich repeated sequence motif, the A repeat, is located in four of five of these regions; the fifth region is also AT rich. The efficiency of viral packaging depends on the number of individual A repeats that are present in the viral genome. The deletion of the entire packaging domain resulted in the loss of virus viability. A virus that contains a multimerized oligonucleotide corresponding to A repeat II in place of the packaging domain could package viral DNA, although with reduced efficiency compared with that of the wild-type virus. Our results also suggest that the spacing of specific sequences at the left end of the Ad5 genome are important for enhancer region function in vivo.  相似文献   

19.
The U(L)15 gene of herpes simplex virus type 1 is composed of two exons. A mutation previously shown to preclude viral DNA cleavage and packaging at the nonpermissive temperature was identified as a change from a highly conserved serine to proline at codon 653. Separate viral mutants that contained stop codons inserted into exon I of U(L)15 (designated S648) or an insertion of the Escherichia coli lacZ gene into a truncated U(L)15 exon II [designated HSV-1(delta U(L)15ExII)] were constructed. Recombinant viruses derived from S648 and HSV-1(delta U(L)15ExII) and containing restored U(L)15 genes were constructed and designated S648R and HSV-1(delta U(L)15ExIIR), respectively. Unlike HSV-1(delta U(L)15ExIIR) and S648R, the viruses containing mutant U(L)15 genes failed to cleave and package viral DNA when propagated on noncomplementing cells. As revealed by electron microscopy, large numbers of enveloped capsids lacking viral DNA accumulated within the cytoplasm of cells infected with either S648 or HSV-1(delta U(L)15ExII) but not in cells infected with HSV-1(delta U(L)15ExIIR) or S648R. Thus, one function of the U(L)15 gene is to effectively prevent immature particles lacking DNA from exiting the nucleus by envelopment at the inner lamella of the nuclear membrane. Cells infected with HSV-1(delta U(L)15ExII) did not express the 75,000- or 35,000-apparent-Mr proteins previously shown to be products of the U(L)15 open reading frame, whereas the 35,000-apparent-Mr protein was readily detectable in cells infected with S648. We conclude that at least the 75,000-Mr protein is required for viral DNA cleavage and packaging and hypothesize that the 35,000-Mr protein is derived from translation of a novel mRNA located partially or completely within the second exon of U(L)15.  相似文献   

20.
Double-stranded RNA (dsRNA) produced during viral infection activates several cellular antiviral responses. Among the best characterized is the shutoff of protein synthesis mediated by the dsRNA-dependent protein kinase (PKR) and the oligoadenylate synthetase (OAS)/RNase L system. As viral replication depends on protein synthesis, many viruses have evolved mechanisms for counteracting the PKR and OAS/RNase L pathways. The murine cytomegalovirus (MCMV) proteins m142 and m143 have been characterized as dsRNA binding proteins that inhibit PKR activation, phosphorylation of the translation initiation factor eIF2α, and a subsequent protein synthesis shutoff. In the present study we analyzed the contribution of the PKR- and the OAS-dependent pathways to the control of MCMV replication in the absence or presence of m142 and m143. We show that the induction of eIF2α phosphorylation during infection with an m142- and m143-deficient MCMV is specifically mediated by PKR, not by the related eIF2α kinases PERK or GCN2. PKR antagonists of vaccinia virus (E3L) or herpes simplex virus (γ34.5) rescued the replication defect of an MCMV strain with deletions of both m142 and m143. Moreover, m142 and m143 bound to each other and interacted with PKR. By contrast, an activation of the OAS/RNase L pathway by MCMV was not detected in the presence or absence of m142 and m143, suggesting that these viral proteins have little or no influence on this pathway. Consistently, an m142- and m143-deficient MCMV strain replicated to high titers in fibroblasts lacking PKR but did not replicate in cells lacking RNase L. Hence, the PKR-mediated antiviral response is responsible for the essentiality of m142 and m143.  相似文献   

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