A helper-dependent system for adenovirus vector production helps define a lower limit for efficient DNA packaging.

Adenoviruses (Ads) are intermediate-sized mammalian DNA viruses with a double-stranded linear genome of 36 kb. The icosohedral virion has been shown to accommodate up to 105% of the wild-type genome length, and genomes larger than this size are either unpackageable or extremely unstable, frequently undergoing DNA rearrangement. Here we show that the Ad virion also has a lower packaging limit of approximately 75% of the wild-type genome length. We have constructed a series of vectors with sizes ranging from 15.1 to 33.6 kb and used these to show that in our Cre/loxP helper-dependent system (R. J. Parks, L. Chen, M. Anton, U. Sankar, M. A. Rudnicki, and F. L. Graham, Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996), vectors with genomes greater than or equal to 27.7 kb are packaged with equal efficiencies, whereas vectors with smaller genomes are inefficiently packaged. A 15.1-kb vector, approximately half the size of the wild-type adenovirus genome, was packaged with an efficiency intermediate between that of the small (21.3- to 25.7-kb) and large (27.7- to 33.5-kb) vectors. Analysis of vector DNA after amplification in helper virus-infected cells showed that vectors below 75% of the Ad genome had undergone DNA rearrangements, whereas larger vectors were unaltered. The 15.1-kb vector was recovered primarily as a mix of head-to-tail and tail-to-tail covalent dimers, with a size of 30 kb. We conclude that the Ad virion can efficiently accommodate viral DNA of greater than 75% of the viral genome but that smaller viral genomes tend to undergo rearrangement, resulting in a final size of greater than approximately 27 kb before they can be efficiently packaged. Knowledge of the lower limit to Ad DNA packaging should allow for the design of better and more stable vectors.     

Mechanisms of cell entry by influenza virus

A wide range of viruses, including many human and animal pathogens representing various taxonomic groups, contain genomes that are enclosed in lipid envelopes. These envelopes are generally acquired in the final stages of assembly, as viruses bud from regions of the membrane of the infected cell at which virally encoded membrane proteins have accumulated. The viruses procure their membranes during this process and mature particles 'pinch off' from the cellular membranes. Under most circumstances, initiation of another round of infection is dependent on two critical functions supplied by the envelope proteins. The virus must bind to cell-surface receptors of a new host cell, and fusion of the viral and cellular membranes must occur to transfer the viral genome into the cell. Enveloped viruses have evolved a variety of mechanisms to execute these two basic functions. Owing to their relative simplicity, studies of binding and fusion using enveloped viruses and their components have contributed significantly to the overall understanding of receptor-ligand interactions and membrane fusion processes - fundamental activities involved in a plethora of biological functions.     

Production of pseudoinfectious yellow fever virus with a two-component genome

Application of genetically modified, deficient-in-replication flaviviruses that are incapable of developing productive, spreading infection is a promising means of designing safe and effective vaccines. Here we describe a two-component genome yellow fever virus (YFV) replication system in which each of the genomes encodes complete sets of nonstructural proteins that form the replication complex but expresses either only capsid or prM/E instead of the entire structural polyprotein. Upon delivery to the same cell, these genomes produce together all of the viral structural proteins, and cells release a combination of virions with both types of genomes packaged into separate particles. In tissue culture, this modified YFV can be further passaged at an escalating scale by using a high multiplicity of infection (MOI). However, at a low MOI, only one of the genomes is delivered into the cells, and infection cannot spread. The replicating prM/E-encoding genome produces extracellular E protein in the form of secreted subviral particles that are known to be an effective immunogen. The presented strategy of developing viruses defective in replication might be applied to other flaviviruses, and these two-component genome viruses can be useful for diagnostic or vaccine applications, including the delivery and expression of heterologous genes. In addition, the achieved separation of the capsid-coding sequence and the cyclization signal in the YFV genome provides a new means for studying the mechanism of the flavivirus packaging process.     

Dimeric RNA Recognition Regulates HIV-1 Genome Packaging

How retroviruses regulate the amount of RNA genome packaged into each virion has remained a long-standing question. Our previous study showed that most HIV-1 particles contain two copies of viral RNA, indicating that the number of genomes packaged is tightly regulated. In this report, we examine the mechanism that controls the number of RNA genomes encapsidated into HIV-1 particles. We hypothesize that HIV-1 regulates genome packaging by either the mass or copy number of the viral RNA. These two distinct mechanisms predict different outcomes when the genome size deviates significantly from that of wild type. Regulation by RNA mass would result in multiple copies of a small genome or one copy of a large genome being packaged, whereas regulation by copy number would result in two copies of a genome being packaged independent of size. To distinguish between these two hypotheses, we examined the packaging of viral RNA that was larger (≈17 kb) or smaller (≈3 kb) than that of wild-type HIV-1 (≈9 kb) and found that most particles packaged two copies of the viral genome regardless of whether they were 17 kb or 3 kb. Therefore, HIV-1 regulates RNA genome encapsidation not by the mass of RNA but by packaging two copies of RNA. To further explore the mechanism that governs this regulation, we examined the packaging of viral RNAs containing two packaging signals that can form intermolecular dimers or intramolecular dimers (self-dimers) and found that one self-dimer is packaged. Therefore, HIV-1 recognizes one dimeric RNA instead of two copies of RNA. Our findings reveal that dimeric RNA recognition is the key mechanism that regulates HIV-1 genome encapsidation and provide insights into a critical step in the generation of infectious viruses.     

Biophysics of viral infectivity: matching genome length with capsid size

In this review, we discuss recent advances in biophysical virology, presenting experimental and theoretical studies on the physical properties of viruses. We focus on the double-stranded (ds) DNA bacteriophages as model systems for all of the dsDNA viruses both prokaryotic and eukaryotic. Recent studies demonstrate that the DNA packaged into a viral capsid is highly pressurized, which provides a force for the first step of passive injection of viral DNA into a bacterial cell. Moreover, specific studies on capsid strength show a strong correlation between genome length, and capsid size and robustness. The implications of these newly appreciated physical properties of a viral particle with respect to the infection process are discussed.     

Synthesis of defective viral DNA in HeLa cells infected with adenovirus type 3.

Virus-specific DNA fragments that are shorter than the full-length viral genomes have been isolated from HeLa cells productively infected with adenovirus type 3. A number of predominant size classes could be detected by gel electrophoresis and hybridization, and the array of sizes was similar or identical to the selection in DNA purified from incomplete particles of this serotype (E. Daniell, J. Virol. 19:685-708, 1976). A large fraction of these short DNA molecules contained long inverted terminal repetitions, as did DNA molecules from incomplete particles. Restriction analysis showed that these subgenomic molecules consist of sequences from the two molecular ends of the normal genome. These results suggest that the predominance of left-hand end fragments seen in packaged incomplete DNAs results from selective packaging, whereas the predominance of certain size classes of intracellular viral DNA is a function of prepackaging events. The incomplete DNAs were generated at all times during viral DNA replication, and the yield relative to complete DNA did not seem to vary significantly with time or multiplicity of infection or when the virus was propagated on different human cell types.     

Vital for Viruses: Intrinsically Disordered Proteins

Viruses infect all kingdoms of life; their genomes vary from DNA to RNA and in size from 2kB to 1 MB or more. Viruses frequently employ disordered proteins, that is, protein products of virus genes that do not themselves fold into independent three-dimensional structures, but rather, constitute a versatile molecular toolkit to accomplish a range of functions necessary for viral infection, assembly, and proliferation. Interestingly, disordered proteins have been discovered in almost all viruses so far studied, whether the viral genome consists of DNA or RNA, and whatever the configuration of the viral capsid or other outer covering. In this review, I present a wide-ranging set of stories illustrating the range of functions of IDPs in viruses. The field is rapidly expanding, and I have not tried to include everything. What is included is meant to be a survey of the variety of tasks that viruses accomplish using disordered proteins.     

DNA Repair Enzyme Uracil DNA Glycosylase Is Specifically Incorporated into Human Immunodeficiency Virus Type 1 Viral Particles through a Vpr-Independent Mechanism

The Vpr protein, encoded by the human immunodeficiency virus type 1 (HIV-1) genome, is one of the nonstructural proteins packaged in large amounts into viral particles. We have previously reported that Vpr associates with the DNA repair enzyme uracil DNA glycosylase (UDG). In this study, we extended these observations by investigating whether UDG is incorporated into virions and whether this incorporation requires the presence of Vpr. Our results, with highly purified viruses, show that UDG is efficiently incorporated either into wild-type virions or into Vpr-deficient HIV-1 virions, indicating that Vpr is not involved in UDG packaging. Using an in vitro protein-protein binding assay, we reveal a direct interaction between the precursor form of UDG and the viral integrase (IN). Finally, we demonstrate that IN-defective viruses fail to incorporate UDG, indicating that IN is required for packaging of UDG into virions.     

Symmetric-strand packaging of recombinant parvovirus LuIII genomes that retain only the terminal regions.

LuIII is an autonomous parvovirus which encapsidates either strand of its genome with similar efficiency in NB324K cells. Two parvoviruses closely related to LuIII, minute virus of mice (MVM) and H-1 virus, encapsidate primarily the minus strand of their genome when grown in the same cell type. It has been postulated that an AT-rich region unique to LuIII is responsible for symmetric encapsidation of plus- and minus-strand genomes by LuIII. To address this hypothesis, recombinant LuIII-luciferase genomes containing or lacking the AT-rich sequence (AT) were packaged into LuIII virions. Hybridization of strand-specific probes to DNA from these virions revealed that either strand of the genome was packaged regardless of the presence of AT. In addition, encapsidation of both strands of the AT+ LuIII-luciferase genome into MVM and H-1 virions was observed, suggesting that MVM and H-1 viral proteins are not responsible for the minus-strand packaging bias of these two viruses. Alignment of the published LuIII and MVMp sequences shows that AT exists as an insertion into an element that, in MVM, binds cellular proteins. We suggest that in LuIII, AT disrupts binding of these cellular proteins, allowing encapsidation of either strand.     

The genome-associated,specific RNA binding proteins of avian and mammalian type C viruses

A structural protein purified from the Rous sarcoma virus (RSV) can specifically bind in vitro to purified avian, but not mammalian, type C viral RNA. Following ultraviolet irradiation of viral particles under conditions which stabilize the polyploid 70S viral RNA, the same polypeptide can be directly purified from the RSV genome. Based on its electrophoretic mobility in polyacrylamide gels containing sodium dodecylsulfate, the RNA binding protein has been identified as the major phosphoprotein (p19) of avian type C viruses. Similar experiments show that the major phosphoproteins of mammalian type C viruses (p12 for murine viruses and p16 for endogenous primate viruses) are also the specific RNA binding proteins and, similarly, are found closely associated with the 70S RNA genomes in the intact viral particles.     

Does a cdc2 Kinase-Like Recognition Motif on the Core Protein of Hepadnaviruses Regulate Assembly and Disintegration of Capsids?

Hepadnaviruses are enveloped viruses, each with a DNA genome packaged in an icosahedral nucleocapsid, which is the site of viral DNA synthesis. In the presence of envelope proteins, DNA-containing nucleocapsids are assembled into virions and secreted, but in the absence of these proteins, nucleocapsids deliver viral DNA into the cell nucleus. Presumably, this step is identical to the delivery of viral DNA during the initiation of an infection. Unfortunately, the mechanisms triggering the disintegration of subviral core particles and delivery of viral DNA into the nucleus are not yet understood. We now report the identification of a sequence motif resembling a serine- or threonine-proline kinase recognition site in the core protein at a location that is required for the assembly of core polypeptides into capsids. Using duck hepatitis B virus, we demonstrated that mutations at this sequence motif can have profound consequences for RNA packaging, DNA replication, and core protein stability. Furthermore, we found a mutant with a conditional phenotype that depended on the cell type used for virus replication. Our results support the hypothesis predicting that this motif plays a role in assembly and disassembly of viral capsids.     

Viral genome methylation as an epigenetic defense against geminiviruses

Geminiviruses encapsidate single-stranded DNA genomes that replicate in plant cell nuclei through double-stranded DNA intermediates that associate with cellular histone proteins to form minichromosomes. Like most plant viruses, geminiviruses are targeted by RNA silencing and encode suppressor proteins such as AL2 and L2 to counter this defense. These related proteins can suppress silencing by multiple mechanisms, one of which involves interacting with and inhibiting adenosine kinase (ADK), a cellular enzyme associated with the methyl cycle that generates S-adenosyl-methionine, an essential methyltransferase cofactor. Thus, we hypothesized that the viral genome is targeted by small-RNA-directed methylation. Here, we show that Arabidopsis plants with mutations in genes encoding cytosine or histone H3 lysine 9 (H3K9) methyltransferases, RNA-directed methylation pathway components, or ADK are hypersensitive to geminivirus infection. We also demonstrate that viral DNA and associated histone H3 are methylated in infected plants and that cytosine methylation levels are significantly reduced in viral DNA isolated from methylation-deficient mutants. Finally, we demonstrate that Beet curly top virus L2- mutant DNA present in tissues that have recovered from infection is hypermethylated and that host recovery requires AGO4, a component of the RNA-directed methylation pathway. We propose that plants use chromatin methylation as a defense against DNA viruses, which geminiviruses counter by inhibiting global methylation. In addition, our results establish that geminiviruses can be useful models for genome methylation in plants and suggest that there are redundant pathways leading to cytosine methylation.     

The phiX174 protein J mediates DNA packaging and viral attachment to host cells

Packaging of viral genomes into their respective capsids requires partial neutralization of the highly negatively charged RNA or DNA. Many viruses, including the Microviridae bacteriophages phiX174, G4, and alpha3, have solved this problem by coding for a highly positively charged nucleic acid-binding protein that is packaged along with the genome. The phiX174 DNA-binding protein, J, is 13 amino acid residues longer than the alpha3 and G4 J proteins by virtue of an additional nucleic acid-binding domain at the amino terminus. Chimeric phiX174 particles containing the smaller DNA-binding protein cannot be generated due to procapsid instability during DNA packaging. However, chimeric alpha3 and G4 phages, containing the phiX174 DNA-binding protein in place of the endogenous J protein, assemble and are infectious, but are less dense than the respective wild-type species. In addition, host cell attachment and native gel migration assays indicate surface variations of these viruses that are controlled by the nature of the J protein. The structure of alpha3 packaged with phiX174 J protein was determined to 3.5A resolution and compared with the previously determined structures of phiX174 and alpha3. The structures of the capsid and spike proteins in the chimeric particle remain unchanged within experimental error when compared to the wild-type alpha3 virion proteins. The amino-terminal region of the phiX174 J protein, which is missing from wild-type alpha3 virions, is mostly disordered in the alpha3 chimera. The differences observed between solution properties of wild-type phiX174, wild-type alpha3, and alpha3 chimera, including their ability to attach to host cells, correlates with the degree of order in the amino-terminal domain of the J protein. When ordered, this domain binds to the interior of the viral capsid and, thus, might control the flexibility of the capsid. In addition, the properties of the phiX174 J protein in the chimera and the results of mutational analyses suggest that an evolutionary correlation may exist between the size of the J protein and the stoichiometry of the DNA pilot protein H, required in the initial stages of infection. Hence, the function of the J protein is to facilitate DNA packaging, as well as to mediate surface properties such as cell attachment and infection.     

Characterization of Empty adenovirus particles assembled in the absence of a functional adenovirus IVa2 protein

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.     

A noninverting genome of a viable herpes simplex virus 1: presence of head-to-tail linkages in packaged genomes and requirements for circularization after infection.

The wild-type herpes simplex virus 1 genome consists of two components, L and S, which invert relative to each other, giving rise to four isomers. Previously we reported the construction of a herpes simplex virus 1 genome, HSV-1(F)I358, from which 15 kilobase pairs of DNA spanning the junction between L and S components were deleted and which no longer inverted (Poffenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2690-2694, 1983). Further studies on the structure of HSV-1(F)I358 revealed the presence of two submolar populations among packaged DNA. The first, comprising no more than 10% of total packaged DNA, consisted of defective genomes with a subunit size of 36 kilobase pairs. The results suggest that this population arose by recombination through a directly repeated sequence inserted in place of the deleted L-S junction. The second minor population consisted of HSV-1(F)I358 DNA linked head-to-tail. Analyses of the structure of HSV-1(F)I358 DNA after infection indicated that the fraction of total DNA linked head-to-tail increased to approximately 40 to 50% within 30 min after exposure of cells to virus. The formation of head-to-tail linkages did not require de novo protein synthesis. Our interpretation of the results is that the termini of full-length DNA molecules are held together during packaging, that a small fraction of the termini is covalently linked during or after packaging, and that the remainder is covalently joined after the release of viral DNA from the infecting virus by either host or viral factors introduced into the cell during infection.     

Computational analysis of DNA replicases in double-stranded DNA viruses: relationship with the genome size

Genome duplication in free-living cellular organisms is performed by DNA replicases that always include a DNA polymerase, a DNA sliding clamp and a clamp loader. What are the evolutionary solutions for DNA replicases associated with smaller genomes? Are there some general principles? To address these questions we analyzed DNA replicases of double-stranded (ds) DNA viruses. In the process we discovered highly divergent B-family DNA polymerases in phiKZ-like phages and remote sliding clamp homologs in Ascoviridae family and Ma-LMM01 phage. The analysis revealed a clear dependency between DNA replicase components and the viral genome size. As the genome size increases, viruses universally encode their own DNA polymerases and frequently have homologs of DNA sliding clamps, which sometimes are accompanied by clamp loader subunits. This pattern is highly non-random. The absence of sliding clamps in large viral genomes usually coincides with the presence of atypical polymerases. Meanwhile, sliding clamp homologs, not accompanied by clamp loaders, have an elevated positive electrostatic potential, characteristic of non-ring viral processivity factors that bind the DNA directly. Unexpectedly, we found that similar electrostatic properties are shared by the eukaryotic 9-1-1 clamp subunits, Hus1 and, to a lesser extent, Rad9, also suggesting the possibility of direct DNA binding.