Roles of LEF-4 and PTP/BVP RNA triphosphatases in processing of baculovirus late mRNAs

The baculovirus Autographa californica nucleopolyhedrovirus encodes two proteins with RNA triphosphatase activity. Late expression factor LEF-4, which is an essential gene, is a component of the RNA polymerase and also encodes the RNA capping enzyme guanylyltransferase. PTP/BVP is also an RNA triphosphatase, but is not essential for viral replication, possibly because its activity is redundant to that of LEF-4. To elucidate the role of these proteins in mRNA cap formation, a mutant virus that lacked both RNA triphosphatase activities was constructed. Infection studies revealed that the double-mutant virus was viable and normal with respect to the production of budded virus. Pulse-labeling studies and immunoblot analyses showed that late gene expression in the double mutant was equivalent to that in the wild type, while polyhedrin expression was slightly reduced. Direct analysis of the mRNA cap structure indicated no alteration of cap processing in the double mutant. Together, these results reveal that baculoviruses replicate and express their late genes at normal levels in the absence of its two different types of RNA triphosphatases.     

Maturation Defects in Temperature-sensitive Mutants of Sindbis Virus

Temperature-sensitive mutants of Sindbis virus, which synthesize viral ribonucleic acid (RNA) but not mature virus at the nonpermissible temperature, were selected for the study of viral maturation. Of these, three mutants which complement each other genetically were used. Two major proteins, the nucleocapsid and membrane proteins, located, respectively, in the viral nucleoid and membrane, were found in intact virions. In cells infected with wild-type Sindbis virus, four distinct types of viral RNA with sedimentation coefficients of 40S, 26S, 20S, and 15S were detected in constant distribution. The 20S RNA was ribonuclease-resistant, whereas the other types were ribonuclease-sensitive. The 40S RNA, identical to that obtained from the virion, was found associated with nucleocapsid protein as a subviral particle, which was assumed to be the nucleoid. Viral materials from cells infected with the mutants under nonpermissive conditions were compared with those from cells infected with wild-type virus, in terms of (i) the distribution of the different types of RNA, (ii) the association of infectious viral RNA into subviral particles, and (iii) the ability of infected cells to hemadsorb goose erythrocytes. According to these criteria, each of the three mutants demonstrated different maturation defects. Defective nucleocapsid proteins and membrane proteins may each account for one of the above mutants. The thrid mutant may have defects in a minor structural protein or possibly a maturation protein which is involved in the assembly of Sindbis virus.     

Hepatitis B virus particle formation in the absence of pregenomic RNA and reverse transcriptase

Cytoplasmic hepatitis B virus (HBV) capsids are not enveloped and secreted unless the packaged RNA pregenome is reverse transcribed. The expression of the capsid protein C, together with envelope proteins in the absence of pregenomic RNA, produced normal amounts of intracellular capsids, but the secretion of virion-like particles was greatly reduced. The I97L C protein mutant, allowing immature nucleocapsid envelopment in the background of an HBV genome, did not promote the envelopment of capsids lacking a pregenome, suggesting that this mutation is not sufficient to induce secretion competence independently of the pregenome.     

Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus.

To identify RNA and protein sequences involved in packaging of human immunodeficiency virus type 1 (HIV-1), various mutations were introduced into the viral genome. Portions of the human immunodeficiency virus type 1 genome between the first splice donor site and the gag initiation codon were deleted to investigate the RNA packaging site (psi). Point mutations that alter cysteine residues in one or both zinc finger motifs of p7, a cleavage product of the gag precursor, were created to study the role of the gag zinc fingers in packaging. The psi site mutants and the gag mutants exhibited similar phenotypes. Cells transfected with the mutant genomes, while expressing normal levels of human immunodeficiency virus type 1 RNA and proteins, produced viral particles that were normal in protein content but lacked detectable viral RNA. These mutant virions were unable to productively infect cells. The combination of human immunodeficiency virus type 1 packaging mutations should minimize fortuitous assembly of infectious virus and may provide a means to produce noninfectious particles for candidate vaccines.     

Mutational analysis of cis-acting RNA signals in segment 7 of influenza A virus

The genomic viral RNA (vRNA) segments of influenza A virus contain specific packaging signals at their termini that overlap the coding regions. To further characterize cis-acting signals in segment 7, we introduced synonymous mutations into the terminal coding regions. Mutation of codons that are normally highly conserved reduced virus growth in embryonated eggs and MDCK cells between 10- and 1,000-fold compared to that of the wild-type virus, whereas similar alterations to nonconserved codons had little effect. In all cases, the growth-impaired viruses showed defects in virion assembly and genome packaging. In eggs, nearly normal numbers of virus particles that in aggregate contained apparently equimolar quantities of the eight segments were formed, but with about fourfold less overall vRNA content than wild-type virions, suggesting that, on average, fewer than eight segments per particle were packaged. Concomitantly, the particle/PFU and segment/PFU ratios of the mutant viruses showed relative increases of up to 300-fold, with the behavior of the most defective viruses approaching that predicted for random segment packaging. Fluorescent staining of infected cells for the nucleoprotein and specific vRNAs confirmed that most mutant virus particles did not contain a full genome complement. The specific infectivity of the mutant viruses produced by MDCK cells was also reduced, but in this system, the mutations also dramatically reduced virion production. Overall, we conclude that segment 7 plays a key role in the influenza A virus genome packaging process, since mutation of as few as 4 nucleotides can dramatically inhibit infectious virus production through disruption of vRNA packaging.     

Rous sarcoma virus nucleic acid-binding protein p12 is necessary for viral 70S RNA dimer formation and packaging.

To study the function(s) of the Rous sarcoma virus nucleic acid-binding protein p12, we constructed mutants by using two restriction sites in the p12 proviral coding sequence of the Prague C strain to insert KpnI synthetic linkers. The two restriction sites are in the same reading frame, which allowed us to construct a deletion mutant lacking the two conserved Cys-His regions and a duplication mutant containing three intact Cys-His boxes. These mutant DNAs were transfected into chicken embryo fibroblasts, and the viral particles produced in a transient assay were characterized biochemically and for infectivity. Our results indicate that the Rous sarcoma virus nucleic acid-binding protein p12 is necessary for genomic RNA packaging but not for particle assembly and is implicated in the formation of a stable 70S dimeric RNA. Moreover, the fact that one mutant was apparently able to package normal 70S RNA but was not infectious suggests a role for p12 during the infection process.     

Noninfectious human immunodeficiency virus type 1 mutants deficient in genomic RNA.

All retroviruses contain, in the nucleocapsid domain of the Gag protein, one or two copies of the sequence Cys-X2-Cys-X4-His-X4-Cys. We have generated a series of mutants in the two copies of this motif present in human immunodeficiency virus type 1. These mutants encoded virus particles that were apparently composed of the normal complement of viral proteins but contained only 2 to 20% of the normal level of genomic RNA. No infectivity could be detected in the mutant particles, while 10(5) infectious U were present in an equivalent amount of wild-type particles. Thus, the mutants have another defect in addition to the inefficiency with which they encapsidate genomic RNA. Our results show that both copies of the motif are required for normal RNA packaging and for infectivity. Mutants of this type may have important applications, including nonhazardous materials for research, immunogens in vaccine and immunotherapy studies, and diagnostic reagents.     

Mapping of functionally important residues of a cysteine-histidine box in the human immunodeficiency virus type 1 nucleocapsid protein.

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein contains two copies of a sequence motif, the cysteine-histidine box, that is conserved among retroviruses. To identify the functionally relevant positions of a cysteine-histidine box, each amino acid in the proximal copy of the motif was individually substituted by site-directed mutagenesis. Mutations at 5 of 14 positions abolished virus replication and reduced the viral RNA content of mutant particles to between 10 and 20% of parental levels. Mutations at other positions had either no or only a minor effect on virus replication and virion RNA content. In vitro binding of RNA to bacterially expressed mutant Pr55gag polyprotein correlated well with the effects of the mutations on particle-associated viral RNA levels. The two different copies of the motif in the HIV-1 nucleocapsid protein are not functionally equivalent, since the conversion of the proximal motif to an exact copy of the distal motif results in a defect in virus replication and a reduction in the viral RNA content of mutant particles. The simultaneous substitution of functionally relevant positions in both motifs led to a significant decline in gag protein export, indicating that the nucleocapsid domain of the gag precursor is also required for efficient assembly or release of the virion.     

Mutational analyses of the influenza A virus polymerase subunit PA reveal distinct functions related and unrelated to RNA polymerase activity

Influenza A viral polymerase is a heterotrimeric complex that consists of PA, PB1, and PB2 subunits. We previously reported that a di-codon substitution mutation (G507A-R508A), denoted J10, in the C-terminal half of PA had no apparent effect on viral RNA synthesis but prevented infectious virus production, indicating that PA may have a novel role independent of its polymerase activity. To further examine the roles of PA in the viral life cycle, we have now generated and characterized additional mutations in regions flanking the J10 site from residues 497 to 518. All tested di-codon mutations completely abolished or significantly reduced viral infectivity, but they did so through disparate mechanisms. Several showed effects resembling those of J10, in that the mutant polymerase supported normal levels of viral RNA synthesis but nonetheless failed to generate infectious viral particles. Others eliminated polymerase activity, in most cases by perturbing the normal nuclear localization of PA protein in cells. We also engineered single-codon mutations that were predicted to pack near the J10 site in the crystal structure of PA, and found that altering residues K378 or D478 each produced a J10-like phenotype. In further studies of J10 itself, we found that this mutation does not affect the formation and release of virion-like particles per se, but instead impairs the ability of those particles to incorporate each of the eight essential RNA segments (vRNAs) that make up the viral genome. Taken together, our analysis identifies mutations in the C-terminal region of PA that differentially affect at least three distinct activities: protein nuclear localization, viral RNA synthesis, and a trans-acting function that is required for efficient packaging of all eight vRNAs.     

Influenza virus assembly and its defects

The most abundant protein within the influenza virus particles is membrane protein (M protein) which forms an inner virus membrane under a lipid bilayer and plays the role of mediator during the process of assembly of a virus particle on plasma membranes. Ehrlich ascites tumor cells (EAT) when infected with influenza virus, strain WSN, produced virus-like particles containing greatly reduced amounts of M protein. Such particles were extremely fragile and easily lost hemagglutinins. The loss of this glycoprotein was accompanied by a decrease in infectious activity.SDS-PAGE analysis of RNA duplexes formed after hybridization of intracellular labeled mRNAs and unlabeled virion RNA showed that the mRNA for M protein was synthesized in EAT nearly in the same amounts as in productively infected chicken fibroblasts. Accordingly, M protein was readily revealed when the polypeptides of infected EAT were analyzed by SDS-PAGE. Thus, the reduced amount of M protein in virus particles was likely not due to the decrease in its synthesis but rather to its defective structure or to its defective transport and misintegration into plasma membranes of EAT.     

Repression of African swine fever virus polyprotein pp220-encoding gene leads to the assembly of icosahedral core-less particles

African swine fever virus (ASFV) polyprotein pp220, encoded by the CP2475L gene, is an N-myristoylated precursor polypeptide that, after proteolytic processing, gives rise to the major structural proteins p150, p37, p34, and p14. These proteins localize at the core shell, a matrix-like virus domain placed between the DNA-containing nucleoid and the inner envelope. In this study, we have examined the role of polyprotein pp220 in virus morphogenesis by means of an ASFV recombinant, v220i, containing an inducible copy of the CP2475L gene regulated by the Escherichia coli repressor-operator system. Under conditions that repress pp220 expression, the virus yield of v220i was about 2.6 log units lower than that of the parental virus or of the recombinant grown under permissive conditions. Electron microscopy revealed that pp220 repression leads to the assembly of icosahedral particles virtually devoid of the core structure. Analysis of recombinant v220i by immunoelectron microscopy, immunoblotting, and DNA hybridization showed that mutant particles essentially lack, besides the pp220-derived products, a number of major core proteins as well as the viral DNA. On the other hand, transient expression of the CP2475L gene in COS cells showed that polyprotein pp220 assembles into electron-dense membrane-bound coats, whereas a mutant nonmyristoylated version of pp220 does not associate with cellular membranes but forms large cytoplasmic aggregates. Together, these findings indicate that polyprotein pp220 is essential for the core assembly and suggest that its myristoyl moiety may function as a membrane-anchoring signal to bind the developing core shell to the inner viral envelope.     

Effects of nucleocapsid mutations on human immunodeficiency virus assembly and RNA encapsidation.

The human immunodeficiency virus (HIV) Pr55Gag precursor proteins direct virus particle assembly. While Gag-Gag protein interactions which affect HIV assembly occur in the capsid (CA) domain of Pr55Gag, the nucleocapsid (NC) domain, which functions in viral RNA encapsidation, also appears to participate in virus assembly. In order to dissect the roles of the NC domain and the p6 domain, the C-terminal Gag protein domain, we examined the effects of NC and p6 mutations on virus assembly and RNA encapsidation. In our experimental system, the p6 domain did not appear to affect virus release efficiency but p6 deletions and truncations reduced the specificity of genomic HIV-1 RNA encapsidation. Mutations in the nucleocapsid region reduced particle release, especially when the p2 interdomain peptide or the amino-terminal portion of the NC region was mutated, and NC mutations also reduced both the specificity and the efficiency of HIV-1 RNA encapsidation. These results implicated a linkage between RNA encapsidation and virus particle assembly or release. However, we found that the mutant ApoMTRB, in which the nucleocapsid and p6 domains of HIV-1 Pr55Gag were replaced with the Bacillus subtilis MtrB protein domain, released particles efficiently but packaged no detectable RNA. These results suggest that, for the purposes of virus-like particle assembly and release, NC can be replaced by a protein that does not appear to encapsidate RNA.     

Murine leukemia virus nucleocapsid mutant particles lacking viral RNA encapsidate ribosomes

A single retroviral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. Gag normally selects the genomic RNA of the virus with high specificity; the nucleocapsid (NC) domain of Gag plays a crucial role in this selection process. However, encapsidation of the viral RNA is completely unnecessary for particle assembly. We previously showed that mutant murine leukemia virus (MuLV) particles that lack viral RNA because of a deletion in the cis-acting packaging signal ("Psi") in the genomic RNA compensate for the loss of the viral RNA by incorporating cellular mRNA. The RNA in wild-type and Psi- particles was also found to be necessary for virion core structure. In the present work, we explored the role of RNA in MuLV particles that lack genomic RNA because of mutations in the NC domain of Gag. Using a fluorescent dye assay, we observed that NC mutant particles contain the same amount of RNA that wild-type virions do. Surprisingly enough, these particles contained large amounts of rRNAs. Furthermore, ribosomal proteins were detected by immunoblotting, and ribosomes were observed inside the particles by electron microscopy. The biological significance of the presence of ribosomes in NC mutant particles lacking genomic RNA is discussed.     

Defective assembly of influenza A virus due to a mutation in the polymerase subunit PA

The RNA-dependent RNA polymerase of influenza A virus is composed of three subunits that together synthesize all viral mRNAs and also replicate the viral genomic RNA segments (vRNAs) through intermediates known as cRNAs. Here we describe functional characterization of 16 site-directed mutants of one polymerase subunit, termed PA. In accord with earlier studies, these mutants exhibited diverse, mainly quantitative impairments in expressing one or more classes of viral RNA, with associated infectivity defects of varying severity. One PA mutant, however, targeting residues 507 and 508, caused only modest perturbations of RNA expression yet completely eliminated the formation of plaque-forming virus. Polymerases incorporating this mutant, designated J10, proved capable of synthesizing translationally active mRNAs and of replicating diverse cRNA or vRNA templates at levels compatible with viral infectivity. Both the mutant protein and its RNA products were appropriately localized in the cytoplasm, where influenza virus assembly occurs. Nevertheless, J10 failed to generate infectious particles from cells in a plasmid-based influenza virus assembly assay, and hemagglutinating material from the supernatants of such cells contained little or no nuclease-resistant genomic RNA. These findings suggest that PA has a previously unrecognized role in assembly or release of influenza virus virions, perhaps influencing core structure or the packaging of vRNAs or other essential components into nascent influenza virus particles.