Genome Encapsidation Research Articles

Mechanism of Reduction in Titers with Vectors Flanked by Chromatin Insulator Elements

Random integration of viral vectors can result in undesirable activation of surrounding genes by enhancers in the vectors (vector genotoxicity), or result in variable expression due to effects of surrounding chromatin on the vector transgene (position effects). Vector genotoxicity has become an area of intense study since the occurrence of gene therapy related leukemias in patients in the French X-SCID trial. Additionally, variability in expression has been shown to compromize therapeutic efficacy in gene therapy for beta-thalassemia, where a high consistent level of expression is necessary. Vectors flanked by the cHS4 insulator, one of the best characterized insulator element, can reduce vector genotoxicity (Neinhuis et al, 2007) and chromatin position effects in γ-retrovirus (Rivella et al, 2000; Yannaki et al, 2002) and lentivirus vectors (Arumugam et al, 2007). Despite these favorable features, the full-length cHS4 insulator, necessary for optimal insulator activity, is infrequently used because it lowers vector infectious titers by 10–15 fold (Ramezani et al, 2003). This reduction in titers is especially limiting with vectors carrying large inserts. We analyzed mechanisms by which this occurred. Insertion of an additional 1.2kb internal cassette to the large human β-globin-LCR (hβ-LCR) lentiviral (LV) vectors did not reduce vector infectious titers. However, insertion of the 1.2Kb cHS4 element reduced titers by more than an order of magntude; showing that reduction in titers by cHS4 was not secondary to a further lengthening of vector genome but this occurred either due to a large insert in the 3′LTR or specific cHS4 sequences that bind cellular factors and hinder viral genome transcription. We then inserted varying sized fragments of cHS4, tandem repeats of the cHS4 core element, or inert DNA spacers in the 3′LTR of the hβ-LCR LV vector, sBG, resulting in vectors with 3′LTR inserts of 0, 250, 400, 500, 650, 800 and 1200bp. After a threshold length of 650bp, infectious titers fell proportional to the size of the insert into the LTR. The same effect was seen with inert DNA spacer elements, showing that this phenomenon was not sequence-specific. We next examined the stage of the vector life-cycle affected by large LTR inserts: lengthening of the 3′LTR did not increase viral readthrough transcription, as measured by northern blot analysis and an enzyme-based assay for readthrough transcription (Higashimoto et al, 2007). Equal amounts of full-length viral genomic transcripts were produced in the packaging cells with vectors with and without the insulator. Similar degree of viral genome encapsidation occurred, as measured by p24 ELISA, virus associated reverse transcriptase and viral RNA analysis, demonstrating that similar amounts of intact viral particles were produced with insulated and uninsulated vector plasmids. However, the insulated vector was inefficiently processed following target cell entry, resulting in less integrated vector; and thus lowers infectious titers. Of note, a vector carrying tandem repeats of the cHS4 core (two 250bp repeats) resulted in increased rate of recombination, with deletion of the insulator core at a high frequency. Thus, we found that large inserts in the viral 3′LTR are packaged efficiently, but have inefficient post-entry viral mRNA processing. These studies have important implications in the design of γ-retrovirus and LV vectors with insulator and other transgene/promoter/enhancer inserts into the LTR.

Unique structures in a tumor herpesvirus revealed by cryo-electron tomography and microscopy

Gammaherpesviruses, including the human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, are causative agents of lymphomas and other malignancies. The structural characterization of these viruses has been limited due to difficulties in obtaining adequate amount of virion particles. Here we report the first three-dimensional structural characterization of a whole gammaherpesvirus virion by an emerging integrated approach of cryo-electron tomography combined with single-particle cryo-electron microscopy, using murine gammaherpesvirus-68 (MHV-68) as a model system. We found that the MHV-68 virion consists of distinctive envelope and tegument compartments, and a highly conserved nucleocapsid. Two layers of tegument are identified: an inner tegument layer tethered to the underlying capsid and an outer, flexible tegument layer conforming to the overlying, pleomorphic envelope, consistent with the sequential viral tegumentation process inside host cells. Surprisingly, comparison of the MHV-68 virion and capsid reconstructions shows that the interactions between the capsid and inner tegument proteins are completely different from those observed in alpha and betaherpesviruses. These observations support the notion that the inner layer tegument across different subfamilies of herpesviruses has evolved significantly to confer specific characteristics related to viral-host interactions, in contrast to a highly conserved capsid for genome encapsidation and protection.

Structural features of the Bluetongue virus NS2 protein

Bluetongue virus (BTV) non-structural protein 2 (NS2) belongs to a class of highly conserved proteins found in members of the orbivirus genus of the reoviridae. NS2 forms large multimeric complexes, localizes to cytoplasmic inclusion bodies in the infected cells and binds non-sequence specifically single-stranded RNA (ssRNA). Due to its ability to bind ssRNA, it has been suggested that the protein is involved in the selection and condensation of the BTV ssRNA segments prior to genome encapsidation. We have previously determined the crystal structure of the 177 amino acid N-terminal domain, sufficient for ssRNA binding ability of NS2, to 2.4Å resolution. The C-terminal domain, as determined at low resolution using small-angle X-ray scattering, is an elongated dimer. This domain expressed in insect cells is phosphorylated at S249 and S259. Electron microscopy of the full-length protein shows a variety of species with the largest having a ring-like appearance. Based on the electron micrographs, the crystal structure of the N-terminal domain and the structure of the C-terminal domain reported here, we propose a model for a decamer of the full-length protein. This decamer changes conformation upon binding of a non-hydrolysable ATP analogue.

A Putative Leucine Zipper within the Herpes Simplex Virus Type 1 UL6 Protein Is Required for Portal Ring Formation

The herpes simplex virus type 1 UL6 protein forms a 12-subunit ring structure at a unique capsid vertex which functions as a conduit for encapsidation of the viral genome. To characterize UL6 protein domains that are involved in intersubunit interactions and interactions with other capsid proteins, we engineered a set of deletion mutants spanning the entire gene. Three deletion constructs, D-5 (Delta 198-295), D-6 (Delta 322-416), and D-LZ (Delta 409-473, in which a putative leucine zipper was removed), were introduced into the viral genome. All three mutant viruses produced only B capsids, indicating a defect in encapsidation. Western blot analysis showed that the UL6 protein was present in the capsids isolated from two mutants, D-6 and D-LZ. The protein encoded by D-5, on the other hand, was not associated with capsids and was instead localized in the cytoplasm of the infected cells, indicating that this deletion affected the nuclear transport of the portal protein. The UL6 protein from the KOS strain (wild type) and the D-6 mutant were purified from insect cells infected with recombinant baculoviruses and shown to form ring structures as assessed by sucrose gradient centrifugation and electron microscopy. In contrast, the D-LZ mutant protein formed aggregates that sedimented throughout the sucrose gradient as a heterogeneous mixture and did not yield stable ring structures. A mutant (L429E L436E) in which two of the heptad leucines of the putative zipper were replaced with glutamate residues also failed to form stable rings. Our results suggest that the integrity of the leucine zipper region is important for oligomer interactions and stable ring formation, which in turn are required for genome encapsidation.

Molecular biology of potexviruses: recent advances

Recent advances in potexvirus research have produced new models describing virus replication, cell-to-cell movement, encapsidation, R gene-mediated resistance and gene silencing. Interactions between distant RNA elements are a central theme in potexvirus replication. The 5' non-translated region (NTR) regulates genomic and subgenomic RNA synthesis and encapsidation, as well as virus plasmodesmal transport. The 3' NTR regulates both plus- and minus-strand RNA synthesis. How the triple gene-block proteins interact for virus movement is still elusive. As the potato virus X (PVX) TGBp1 protein gates plasmodesmata, regulates virus translation and is a suppressor of RNA silencing, further research is needed to determine how these properties contribute to propelling virus through the plasmodesmata. Specifically, TGBp1 suppressor activity is required for virus movement, but how the silencing machinery relates to plasmodesmata is not known. The TGBp2 and TGBp3 proteins are endoplasmic reticulum (ER)-associated proteins required for virus movement. TGBp2 associates with ER-derived vesicles that traffic along the actin network. Future research will determine whether the virus-induced vesicles are cytopathic structures regulating events along the ER or are vehicles carrying virus to the plasmodesmata for transfer into neighbouring cells. Efforts to assemble virions in vitro identified a single-tailed particle (STP) comprising RNA, coat protein (CP) and TGBp1. It has been proposed that TGBp1 aids in transport of virions or STP between cells and ensures translation of RNA in the receiving cells. PVX is also a tool for studying Avr-R gene interactions and gene silencing in plants. The PVX CP is the elicitor for the Rx gene. Recent reports of the PVX CP reveal how CP interacts with the Rx gene product.

A host cell membrane protein, golgin-97, is essential for poxvirus morphogenesis

Acquisition of the membrane and genome encapsidation is an important step in the replication of enveloped viruses. The biogenesis of the poxviral primary membrane and the core as well as the mechanisms of their maturation are poorly understood. Using RNA interference approach, we demonstrate that a cellular trans-Golgi network membrane protein, golgin-97, is essential for virus replication. Analysis of the virion morphology in the cells depleted of golgin-97 shows that the protein is required for the virus morphogenesis and, in particular, for the formation of the first infectious virus form, mature virus, but not its precursor, immature virus. This suggests that golgin-97 may be involved in the maturation of the virus core and, potentially, the virus membrane.

Effect of cAMP-dependent protein kinase A (PKA) on HCV nucleocapsid assembly and degradation

The primary function of the hepatitis C virus (HCV) core protein is genome encapsidation. Core protein is also subject to post-translational modifications that can impact on the assembly process. In this report, we have studied the effect of cAMP-dependent protein kinase A (PKA) phosphorylation on its assembly and stability in a yeast Pichia pastoris expression system. We have recently shown that co-expression of the human signal peptide peptidase and core protein (amino acids 1-191) in yeast leads to the formation of nucleocapsid-like particles (NLPs) that are morphologically similar to the wild-type HCV capsid. In this system, we expressed mutants S53A and S116A and mutants S53D and S116D to abolish or mimic PKA phosphorylation, respectively. None of these mutations affected HCV assembly, but S116D led to the degradation of core protein. We also showed that nonenveloped NLPs were labelled in vitro by PKA, suggesting that the phosphorylation sites are available at the surface of the NLPs. The co-expression of human PKA with core and human signal peptide peptidase in yeast did not produce phosphorylated NLPs and led to a decreased accumulation of nonenveloped particles. Mutation S116A restored the core protein content. These results suggest that PKA phosphorylation can modulate HCV core levels in infected cells.

Self-Complementary Recombinant Adeno-Associated Viral Vectors: Packaging Capacity And The Role of Rep Proteins in Vector Purity

Self-complementary adeno-associated viral (scAAV) vectors bypass the requirement for viral second-strand DNA synthesis, but the packaging capacity of these vectors ( approximately 2.4 kb) is significantly smaller than that of conventional AAV vectors ( approximately 4.8 kb). We constructed human recombinant green fluorescent protein (hrGFP) expression cassettes ranging from 2.3 to 4.1 kb. Each vector was biologically active, but the transduction efficiency of vectors containing <3.3-kb genomes was significantly higher than those containing 3.5-kb genomes or larger. However, scAAV vectors containing up to approximately 3.3-kb genomes also contained single-stranded genomes, and 3.5-kb and larger genomes were packaged only as single-stranded DNA. These data suggest that the maximum packaging capacity of scAAV vectors is approximately 3.3 kb. The production of single-stranded genomes was not due to repair of the terminal resolution site (trs) in the inverted terminal repeats in the AAV genome, but rather was partly due to the use of AAV helper plasmid, known to lead to higher levels of expression of Rep proteins. The use of a helper plasmid known to lead to reduced levels of Rep proteins led to the generation of scAAV vectors that contained approximately 90% of the viral genomes in double-stranded forms. These studies demonstrate the feasibility of achieving encapsidation of larger genomes into scAAV vectors than was suggested originally, but underscore the need to exercise caution in using the appropriate helper plasmid to generate scAAV stocks capable of high-efficiency transduction that are relatively free of single-stranded DNA-containing vectors.

A Two-hybrid Screen Identifies Cathepsins B and L as Uncoating Factors for Adeno-associated Virus 2 and 8

Vectors based on different serotypes of adeno-associated virus hold great promise for human gene therapy, based on their unique tissue tropisms and distinct immunological profiles. A particularly interesting candidate is AAV8, which can efficiently and rapidly transduce a wide range of tissues in vivo. To further unravel the mechanisms behind AAV8 transduction, we used yeast two-hybrid analyses to screen a mouse liver complementary DNA library for cellular proteins capable of interacting with the viral capsid proteins. In total, we recovered approximately 700 clones, comprising over 300 independent genes. Sequence analyses revealed multiple hits for over 100 genes, including two encoding the endosomal cysteine proteases cathepsins B and L. Notably, these two proteases also physically interacted with the corresponding portion of the AAV2 capsid in yeast, but not with AAV5. We demonstrate that cathepsins B and L are essential for efficient AAV2- and AAV8-mediated transduction of mammalian cells, and document the ability of purified cathepsin B and L proteins to bind and cleave intact AAV2 and AAV8 particles in vitro. These data suggest that cathepsin-mediated cleavage could prime AAV capsids for subsequent nuclear uncoating, and indicate that analysis of additional genes recovered in our screen may help to further elucidate the mechanisms behind transduction by AAV8 and related serotypes.

An Extended Stem-Loop 1 Is Necessary for Human Immunodeficiency Virus Type 2 Replication and Affects Genomic RNA Encapsidation

Genomic RNA encapsidation in lentiviruses is a highly selective and regulated process. The unspliced RNA molecules are selected for encapsidation from a pool of many different viral and cellular RNA species. Moreover, two molecules are encapsidated per viral particle, where they are found associated as a dimer. In this study, we demonstrate that a 10-nucleotide palindromic sequence (pal) located at the 3' end of the psi encapsidation signal is critical for human immunodeficiency virus type 2 (HIV-2) replication and affects genomic RNA encapsidation. We used short-term and long-term culture of pal-mutated viruses in permissive C8166 cells and their phenotypic reversion to show the existence of a structurally extended SL1 during HIV-2 replication, formed by the interaction of the 3' end of the pal within psi with a motif located downstream of SL1. The stem extending HIV-2 SL1 is structurally similar to stem B described for HIV-1 SL1. Despite the high degree of phylogenetic conservation, these results show that mutant viruses are viable when the autocomplementary nature of the pal sequence is disrupted, but not without a stable stem B. Our observations show that formation of the extended SL1 is necessary during viral replication and positively affects HIV-2 genomic RNA encapsidation. Sequestration of part of the packaging signal into SL1 may be a means of regulating its presentation during the replication cycle.

Constraints to Genetic Exchange Support Gene Coadaptation in a Tripartite RNA Virus

Genetic exchange by recombination, or reassortment of genomic segments, has been shown to be an important process in RNA virus evolution, resulting often in important phenotypic changes affecting host range and virulence. However, data from numerous systems indicate that reassortant or recombinant genotypes could be selected against in virus populations and suggest that there is coadaptation among viral genes. Little is known about the factors affecting the frequency of reassortants and recombinants along the virus life cycle. We have explored this issue by estimating the frequency of reassortant and recombinant genotypes in experimental populations of Cucumber mosaic virus derived from mixed infections with four different pairs of isolates that differed in about 12% of their nucleotide sequence. Genetic composition of progeny populations were analyzed at various steps of the virus life cycle during host colonization: infection of leaf cells, cell-to-cell movement within the inoculated leaf, encapsidation of progeny genomes, and systemic movement to upper noninoculated leaves. Results indicated that reassortant frequencies do not correspond to random expectations and that selection operates against reassortant genotypes. The intensity of selection, estimated through the use of log-linear models, increased as host colonization progressed. No recombinant was detected in any progeny. Hence, results showed the existence of constraints to genetic exchange linked to various steps of the virus life cycle, so that genotypes with heterologous gene combinations were less fit and disappeared from the population. These results contribute to explain the low frequency of recombinants and reassortants in natural populations of many viruses, in spite of high rates of genetic exchange. More generally, the present work supports the hypothesis of coadaptation of gene complexes within the viral genomes.

Human Immunodeficiency Virus Type 1 Gag Polyprotein Modulates Its Own Translation

The full-length viral RNA of human immunodeficiency virus type 1 (HIV-1) functions both as the mRNA for the viral structural proteins Gag and Gag/Pol and as the genomic RNA packaged within viral particles. The packaging signal which Gag recognizes to initiate genome encapsidation is in the 5' untranslated region (UTR) of the HIV-1 RNA, which is also the location of translation initiation complex formation. Hence, it is likely that there is competition between the translation and packaging processes. We studied the ability of Gag to regulate translation of its own mRNA. Gag had a bimodal effect on translation from the HIV-1 5' UTR, stimulating translation at low concentrations and inhibiting translation at high concentrations in vitro and in vivo. The inhibition was dependent upon the ability of Gag to bind the packaging signal through its nucleocapsid domain. The stimulatory activity was shown to depend on the matrix domain of Gag. These results suggest that Gag controls the equilibrium between translation and packaging, ensuring production of enough molecules of Gag to make viral particles before encapsidating its genome.

The Structural and Functional Role of RNA in Icosahedral Virus Assembly

Despite tremendous advances in high-resolution structure determination of virus particles, the organization of encapsidated genomes and their role during assembly are poorly understood. This article summarizes recent insights from structural, biochemical, and genetic analyses of icosahedral viruses that contain single-stranded, positive-sense RNA genomes. X-ray crystallography of several viruses in this category has provided tantalizing glimpses of portions of the packaged nucleic acid, contributing crucial information on how the genome might be folded within the virion. This information combined with theoretical considerations and data from molecular approaches suggests mechanisms by which coat proteins interact with genomic RNA to shape it into a conformation that is compatible with the geometry of the virion. It appears that RNA, in addition to its function as a repository for genetic information, plays an important structural role during assembly and can on occasion override the ability of the coat protein to form a particle with defined icosahedral symmetry.

Encapsidation Determinants Located Downstream of the Major Splice Donor in the Maedi-Visna Virus Leader Region

We investigated the role of the 5'-untranslated region between the primer binding site and the gag initiation codon in ovine lentivirus maedi-visna virus (MVV) genomic RNA encapsidation. We identified five computer-predicted stem-loops, three of which were highly conserved in primary sequence and structure. One stable 83-nucleotide (nt) stem-loop (SL4) was not conserved in the primary sequence, but phylogenetic analysis revealed several base pair covariations. The deletion of individual stem-loops did not markedly affect the relative encapsidation efficiency (REE). Only one mutant, carrying a disruption of a 31-nt stem-loop (SL5), had 58% REE in fetal ovine synovial (FOS) cells. A 168-nt deletion (Delta3MSD) downstream of the major splice donor (MSD) which removed three stem-loops, including SL5, resulted in 24% and 20% REE in FOS and 293T cells, respectively. A 100-nt deletion (Delta5MSD) upstream of the MSD resulted in 15-fold lower cellular genomic RNA levels than the wild-type levels in 293T cells. The Delta5MSD mutant and a double mutant (DM) (Delta5MSD and Delta3MSD) did not express detectable levels of virion proteins in 293T cells. In contrast, the region deleted in Delta5MSD was dispensable in FOS cells, and the DM had the same REE as the Delta3MSD virus. Thus, the region upstream of the MSD contains sequences critical for RNA and protein expression in a cell type-specific fashion. Our results indicate that MVV encapsidation determinants are located downstream of the MSD. These results provide comparative insight into lentiviral encapsidation and can be utilized in the design of MVV-based gene transfer vectors.

Caliciviruses Differ in Their Functional Requirements for eIF4F Components

Two classes of viruses, namely members of the Potyviridae and Caliciviridae, use a novel mechanism for the initiation of protein synthesis that involves the interaction of translation initiation factors with a viral protein covalently linked to the viral RNA, known as VPg. The calicivirus VPg proteins can interact directly with the initiation factors eIF4E and eIF3. Translation initiation on feline calicivirus (FCV) RNA requires eIF4E because it is inhibited by recombinant 4E-BP1. However, to date, there have been no functional studies carried out with respect to norovirus translation initiation, because of a lack of a suitable source of VPg-linked viral RNA. We have now used the recently identified murine norovirus (MNV) as a model system for norovirus translation and have extended our previous studies with FCV RNA to examine the role of the other eIF4F components in translation initiation. We now demonstrate that, as with FCV, MNV VPg interacts directly with eIF4E, although, unlike FCV RNA, translation of MNV RNA is not sensitive to 4E-BP1, eIF4E depletion, or foot-and-mouth disease virus Lb protease-mediated cleavage of eIF4G. We also demonstrate that both FCV and MNV RNA translation require the RNA helicase component of the eIF4F complex, namely eIF4A, because translation was sensitive (albeit to different degrees) to a dominant negative form and to a small molecule inhibitor of eIF4A (hippuristanol). These results suggest that calicivirus RNAs differ with respect to their requirements for the components of the eIF4F translation initiation complex.

Phosphorylation of hepatitis B virus Cp at Ser87 facilitates core assembly.

Protein-protein interactions can be regulated by protein modifications such as phosphorylation. Some of the phosphorylation sites (Ser155, Ser162 and Ser170) of HBV (hepatitis B virus) Cp have been discovered and these sites are implicated in the regulation of viral genome encapsidation, capsid localization and nucleocapsid maturation. In the present report, the dimeric form of HBV Cp was phosphorylated by PKA (protein kinase A), but not by protein kinase C in vitro, and the phosphorylation of dimeric Cp facilitated HBV core assembly. Matrix-assisted laser-desorption ionization-time-of-flight analysis revealed that the HBV Cp was phosphorylated at Ser87 by PKA. This was further confirmed using a mutant HBV Cp with S87G mutation. The S87G mutation inhibited the phosphorylation and, as a result, the in vitro HBV core assembly was not facilitated by PKA. In addition, when either pCMV/FLAG-Core(WT) or pCMV/FLAG-Core(S87G) was transfected into HepG2 cells, few mutant Cps (S87G) assembled into capsids compared with the wild-type (WT) Cps, although the same level of total Cps was expressed in both cases. In conclusion, PKA facilitates HBV core assembly through phosphorylation of the HBV Cp at Ser87.

Modulation of the Viral ATPase Activity by the Portal Protein Correlates with DNA Packaging Efficiency

DNA packaging in tailed bacteriophages and herpesviruses requires assembly of a complex molecular machine at a specific vertex of a preformed procapsid. As in all these viruses, the DNA translocation motor of bacteriophage SPP1 is composed of the portal protein (gp6) that provides a tunnel for DNA entry into the procapsid and of the viral ATPase (gp1-gp2 complex) that fuels DNA translocation. Here we studied the cross-talk between the components of the motor to control its ATP consumption and DNA encapsidation. We showed that gp6 embedded in the procapsid structure stimulated more than 10-fold the gp2 ATPase activity. This stimulation, which was significantly higher than the one conferred by isolated gp6, depended on the presence of gp1. Mutations in different regions of gp6 abolished or decreased the gp6-induced stimulation of the ATPase. This effect on gp2 activity was observed both in the presence and in the absence of DNA and showed a strict correlation with the efficiency of DNA packaging into procapsids containing the mutant portals. Our results demonstrated that the portal protein has an active control over the viral ATPase activity that correlates with the performance of the DNA packaging motor.

The Capsid-Associated UL25 Protein of the Alphaherpesvirus Pseudorabies Virus Is Nonessential for Cleavage and Encapsidation of Genomic DNA but Is Required for Nuclear Egress of Capsids

Homologs of the UL25 gene product of herpes simplex virus (HSV) have been identified in all three subfamilies of the Herpesviridae. However, their exact function during viral replication is not yet known. Whereas earlier studies indicated that the UL25 protein of HSV-1 is not required for cleavage of newly replicated viral DNA but is necessary for stable encapsidation (A. R. McNab, P. Desai, S. Person, L. Roof, D. R. Thompson, W. W. Newcomb, J. C. Brown, and F. L. Homa, J. Virol. 72:1060-1070, 1998), viral DNA packaging has recently been demonstrated to occur in the absence of UL25, although at significantly decreased levels compared to wild-type HSV-1 (N. Stow, J. Virol. 75:10755-10765 2001). To clarify the functional role of UL25 we analyzed the homologous protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL25 was found to be essential for viral replication, as a mutant virus lacking the UL25 protein required UL25-expressing cells for productive propagation. In the absence of the UL25 protein, newly replicated PrV DNA was cleaved and DNA-containing C-type capsids were detected in infected cell nuclei. However, although capsids were frequently found in close association with the inner nuclear membrane, nuclear egress was not observed. Consequently, no capsids were found in the cytoplasm, resulting in an inhibition of virion morphogenesis. In contrast, the formation of capsidless enveloped tegument structures (L particles) in the cytoplasm was readily observed. Thus, our data demonstrate that the PrV UL25 protein is not essential for cleavage and encapsidation of viral genomes, although both processes occur more efficiently in the presence of the protein. However, the presence of the PrV UL25 protein is a prerequisite for nuclear egress. By immunoelectron microscopy, we detected UL25-specific label on DNA-containing C capsids but not on other intranuclear immature or defective capsid forms. Thus, the PrV UL25 protein may represent the hitherto missing trigger that allows primary envelopment preferably of DNA-filled C capsids.

Polydnavirus genomes reflect their dual roles as mutualists and pathogens

Symbionts often exhibit significant reductions in genome complexity while pathogens often exhibit increased complexity through acquisition and diversification of virulence determinants. A few organisms have evolved complex life cycles in which they interact as symbionts with one host and pathogens with another. How the predicted and opposing influences of symbiosis and pathogenesis affect genome evolution in such instances, however, is unclear. The Polydnaviridae is a family of double-stranded (ds) DNA viruses associated with parasitoid wasps that parasitize other insects. Polydnaviruses (PDVs) only replicate in wasps but infect and cause severe disease in parasitized hosts. This disease is essential for survival of the parasitoid's offspring. Thus, a true mutualism exists between PDVs and wasps as viral transmission depends on parasitoid survival and parasitoid survival depends on viral infection of the wasp's host. To investigate how life cycle and ancestry affect PDVs, we compared the genomes of Campoletis sonorensis ichnovirus ( CsIV) and Microplitis demolitor bracovirus ( MdBV). CsIV and MdBV have no direct common ancestor, yet their encapsidated genomes share several features including segmentation, diversification of virulence genes into families, and the absence of genes required for replication. In contrast, CsIV and MdBV share few genes expressed in parasitized hosts. We conclude that the similar organizational features of PDV genomes reflect their shared life cycle but that PDVs associated with ichneumonid and braconid wasps have likely evolved different strategies to cause disease in the wasp's host and promote parasitoid survival.

Single Point Mutations in the Zinc Finger Motifs of the Human Immunodeficiency Virus Type 1 Nucleocapsid Alter RNA Binding Specificities of the Gag Protein and Enhance Packaging and Infectivity

A specific interaction between the nucleocapsid (NC) domain of the Gag polyprotein and the RNA encapsidation signal (Psi) is required for preferential incorporation of the retroviral genomic RNA into the assembled virion. Using the yeast three-hybrid system, we developed a genetic screen to detect human immunodeficiency virus type 1 (HIV-1) Gag mutants with altered RNA binding specificities. Specifically, we randomly mutated full-length HIV-1 Gag or its NC portion and screened the mutants for an increase in affinity for the Harvey murine sarcoma virus encapsidation signal. These screens identified several NC zinc finger mutants with altered RNA binding specificities. Furthermore, additional zinc finger mutants that also demonstrated this phenotype were made by site-directed mutagenesis. The majority of these mutants were able to produce normal virion-like particles; however, when tested in a single-cycle infection assay, some of the mutants demonstrated higher transduction efficiencies than that of wild-type Gag. In particular, the N17K mutant showed a seven- to ninefold increase in transduction, which correlated with enhanced vector RNA packaging. This mutant also packaged larger amounts of foreign RNA. Our results emphasize the importance of the NC zinc fingers, and not other Gag sequences, in achieving specificity in the genome encapsidation process. In addition, the described mutations may contribute to our understanding of HIV diversity resulting from recombination events between copackaged viral genomes and foreign RNA.