Non-random deletions in human foamy virus long terminal repeat during viral infection

We have characterized a new form of human foamy virus (HFV) non-random deleted long terminal repeat (LTR) sizing 1078-bp, deleted in its U3 region, sensitive to the viral transactivator and functional in an infectious proviral clone. Besides two known HFV LTRs of 1260-bp and 1123-bp, this LTR represents the smallest, designed S. Analysis of the LTR sequence shows the presence of short direct repeats surrounding the deletions, suggesting a mechanism generating deletion by misalignment of the growing strand during replication. Our data suggest that the deleted LTRs, preferentially associated with chronic viral infection, could be related with viral persistence.     

Infectious proviral clones of chimpanzee foamy virus (SFVcpz) generated by long PCR reveal close functional relatedness to human foamy virus

Infectious proviral clones of simian foamy virus isolated from chimpanzee (SFVcpz) were generated by long PCR. Two overlapping fragments representing the complete provirus were amplified from genomic DNA of infected cells. Four 8.8-kbp amplimers extending from base 1 of the provirus into the env gene and five 4.45-kbp amplimers reaching from env to the end of the 3'-LTR were cloned into pCR II. Subsequently, the proviral fragments were combined in a chessboard manner to generate 20 plasmids containing full-length proviral DNA. Four plasmids produced infectious virus after transfection of susceptible cells. A distinct proviral form bearing a deletion in the transactivator gene joining both exons of a second regulatory gene present in wild-type foamy virus-infected cells started to emerge 48 hr after transfection of BHK cells with infectious SFVcpz DNA. This observation supports a novel hypothesis to explain establishment of foamy virus latency. The transactivator protein Taf of SFVcpz transcomplemented for the homologous protein Bel-1 of the unique human foamy virus isolate (HFV) and Bel-1 exhibited the reciprocal activity, suggesting that HFV could represent a variant of chimpanzee foamy virus.     

An evolutionarily conserved splice generates a secreted env-Bet fusion protein during human foamy virus infection

Foamy viruses (spumaretroviruses) represent a retroviral genus which exhibits unusual features relating it to pararetroviruses. Previously, we reported the existence of a protein species harboring Env, Bel, and Bet epitopes in human foamy virus (HFV)-infected cells (M. L. Giron, F. Rozain, M. C. Debons-Guillemin, M. Canivet, J. Périès, and R. Emanoil-Ravier, J. Virol. 67:3596-3600, 1993). Here, we identify this protein as a 160-kDa Env-Bet fusion glycoprotein (gp160) translated from an mRNA species harboring a highly conserved splice site which deletes the membrane anchor domain of Env and fuses the env open reading frame with that of bel1/bet. While gp160 and Bet proteins were both secreted into the supernatant, only Bet was taken up by recipient cells. Since Bet plays a key role in the switch from lytic to chronic infection, secretion of Bet and gp160, together with cellular uptake of Bet, could be highly relevant for both immune response and development of HFV infection in vivo.     

The a sequence is dispensable for isomerization of the herpes simplex virus type 1 genome

The herpes simplex virus type 1 (HSV-1) genome consists of two components, L (long) and S (short), that invert relative to each other during productive infection to generate four equimolar isomeric forms of viral DNA. Recent studies have indicated that this genome isomerization is the result of DNA replication-mediated homologous recombination between the large inverted repeat sequences that exist in the genome, rather than site-specific recombination through the terminal repeat a sequences present at the L-S junctions. However, there has never been an unequivocal demonstration of the dispensability of the latter element for this process using a recombinant virus whose genome lacks a sequences at its L-S junctions. This is because the genetic manipulations required to generate such a viral mutant are not possible using simple marker transfer, since the cleavage and encapsidation signals of the a sequence represent essential cis-acting elements which cannot be deleted outright from the viral DNA. To circumvent this problem, a simple two-step strategy was devised by which essential cis-acting sites like the a sequence can be readily deleted from their natural loci in large viral DNA genomes. This method involved initial duplication of the element at a neutral site in the viral DNA and subsequent deletion of the element from its native site. By using this approach, the a sequence at the L-S junction was rendered dispensable for virus replication through the insertion of a second copy into the thymidine kinase (TK) gene of the viral DNA; the original copies at the L-S junctions were then successfully deleted from this virus by conventional marker transfer. The final recombinant virus, HSV-1::L-S(delta)a, was found to be capable of undergoing normal levels of genome isomerization on the basis of the presence of equimolar concentrations of restriction fragments unique to each of the four isomeric forms of the viral DNA. Interestingly, only two of these genomic isomers could be packaged into virions. This restriction was the result of inversion of the L component during isomerization, which prevented two of the four isomers from having the cleavage and encapsidation signals of the a sequence in the TK gene in a packageable orientation. This phenomenon was exploited as a means of directly measuring the kinetics of HSV-1::L-S(delta)a genome isomerization. Following infection with virions containing just the two packaged genomic isomers, all four isomers were readily detected at a stage in infection coincident with the onset of DNA replication, indicating that the loss of the a sequence at the L-S junction had no adverse effect on the frequency of isomerization events in this virus. These results therefore validate the homologous recombination model of HSV-1 genome isomerization by directly demonstrating that the a sequence at the L-S junction is dispensable for this process. The strategy used to remove the a sequence from the HSV-1 genome in this work should be broadly applicable to studies of essential cis-acting elements in other large viral DNA molecules.     

Compensatory point mutations in the human immunodeficiency virus type 1 Gag region that are distal from deletion mutations in the dimerization initiation site can restore viral replication

The dimerization initiation site (DIS), downstream of the long terminal repeat within the human immunodeficiency virus type 1 (HIV-1) genome, can form a stem-loop structure (SL1) that has been shown to be involved in the packaging of viral RNA. In order to further determine the role of this region in the virus life cycle, we deleted the 16 nucleotides (nt) at positions +238 to +253 within SL1 to generate a construct termed BH10-LD3 and showed that this virus was impaired in viral RNA packaging, viral gene expression, and viral replication. Long-term culture of these mutated viruses in MT-2 cells, i.e., 18 passages, yielded revertant viruses that possessed infectivities similar to that of the wild type. Cloning and sequencing showed that these viruses retained the original 16-nt deletion but possessed two additional point mutations, which were located within the p2 and NC regions of the Gag coding region, respectively, and which were therefore named MP2 and MNC. Site-directed mutagenesis studies revealed that both of these point mutations were necessary to compensate for the 16-nt deletion in BH10-LD3. A construct with both the 16-nt deletion and the MP2 mutation, i.e., LD3-MP2, produced approximately five times more viral protein than BH10-LD3, while the MNC mutation, i.e., construct LD3-MNC, reversed the defects in viral RNA packaging. We also deleted nt +261 to +274 within the 3' end of SL1 and showed that the diminished infectivity of the mutated virus, termed BH10-LD4, could also be restored by the MP2 and MNC point mutations. Therefore, compensatory mutations within the p2 and NC proteins, distal from deletions within the DIS region of the HIV genome, can restore HIV replication, viral gene expression, and viral RNA packaging to control levels.     

Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE) functions in a position-dependent manner

The Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE) is a cis-acting RNA element located in the 3' untranslated region (UTR) of the viral genome. The HIV-1 and SIV Rev/RRE regulatory system can be replaced with MPMV CTE (Bray et al., 1994; Zolotukhin et al., 1994; Rizvi et al., 1996a); similarly, CTE function can also be replaced by the HIV or SIV Rev/RRE regulatory system (Rizvi et al., 1996b; Ernst et al., 1997). In addition, we have shown that in the context of the SIV genome, position is important for CTE function (Rizvi et al., 1996a). To determine the importance of position for CTE function in the context of the MPMV genome, MPMV molecular clones were generated by deleting CTE or removing it from the 3' UTR and placing it in the approximately 40 bp of intervening sequences between the pol termination codon and env initiation codon. A test of these molecular clones in a single round of replication assay revealed that deletion or displacement of CTE in the intervening sequences between pol and env completely abrogated virus replication. Western blot analysis of cell lysates and pelleted culture supernatants revealed negligible amounts of Pr78 Gag/Pol precursor and the processed p27(gag) when CTE was deleted or displaced. Slot blot analysis of fractionated RNAs revealed entrapment of the viral Gag/Pol mRNA in the nucleus with CTE deletion or displacement. Upon reinsertion of CTE in the original genomic position of clones with the deleted or displaced CTE, virus replication, Gag/Pol protein production, and nucleocytoplasmic transport of viral mRNA were restored to normal levels. Displacement of CTE to the 5' UTR immediately upstream of the Gag initiation codon also resulted in aberrant Gag/Pol protein production and nucleocytoplasmic transport of viral RNA. Reinsertion of CTE at the original genomic position of the clone with CTE displacement at the 5' UTR restored normal Gag/Pol protein production and RNA transport, demonstrating that the 3' terminal position of CTE is important for its function. To explore why the 3' terminal location of CTE is important, heterologous DNA sequences of increasing lengths were inserted between CTE and the polyadenylation (poly(A)) signal of the virus to augment the distance between the two cis-acting elements. Test of these constructs revealed that CTE function was progressively lost with incremental increase in distance between CTE and poly(A). To explore this relationship further, CTE was displaced to the env region approximately 2000 bp upstream of the poly(A) signal which abrogated CTE function. However, cloning of poly(A) signal to approximately 200 bp downstream of CTE in the env region (the natural distance between CTE and poly(A)) restored CTE function. Together, these results demonstrate that the close proximity of CTE to the poly(A) signal is important for CTE function, suggesting a functional interaction between CTE and the polyadenylation machinery.     

Cells expressing the human foamy virus (HFV) accessory Bet protein are resistant to productive HFV superinfection

Bet is a foamy virus (FV) accessory protein not required for virus replication. The function of Bet is not understood. We report on the generation of cell lines stably expressing the HFV Bet protein. In Bet+ cells, HFV replication was reduced by approximately 3-4 orders of magnitude compared with control cells. The HFV Bet-expressing cells only partially resisted infection by the distantly related feline FV (FFV). Pseudotyping experiments, using murine retroviral vectors with an HFV envelope, revealed that the resistance was not due to downregulation of the unknown HFV receptor. In transfection experiments, using proviral reporter gene constructs and infectious proviruses, no significant differences were detected between Bet+ and control cells. In infection experiments, HFV vectors expressing an indicator gene under control of the HFV promoters showed no activity in Bet+ cells. The results are best compatible with the hypothesis that the main block to productive superinfection of Bet+ cells occurs at an early stage of replication between virus entry and provirus establishment. We suggest that inhibition of provirus integration by Bet protein may serve a distinct function in the unique foamy virus replication cycle.     

Persistent baculovirus infection results from deletion of the apoptotic suppressor gene p35

Infection with the wild-type baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) results in complete death of Spodoptera frugiperda (Sf) cells. However, infection of Sf cells with AcMNPV carrying a mutation or deletion of the apoptotic suppressor gene p35 allowed the cloning of surviving Sf cells that harbored persistent viral genomes. Persistent infection established with the virus with p35 mutated or deleted was blocked by stable transfection of p35 in the host genome or by insertion of the inhibitor of apoptosis (iap) gene into the viral genome. These artificially established persistently virus-infected cells became resistant to subsequent viral challenge, and some of the cell lines carried large quantities of viral DNA capable of early gene expression. Continuous release of viral progenies was evident in some of the persistently virus-infected cells, and transfection of p35 further stimulated viral activation of the persistent cells, including the reactivation of viruses in those cell lines without original continuous virus release. These results have demonstrated the successful establishment of persistent baculovirus infections under laboratory conditions and that their establishment may provide a novel continuous, nonlytic baculovirus expression system in the future.     

Directed integration of minute virus of mice DNA into episomes

Recent studies with adeno-associated virus (AAV) have shown that site-specific integration is directed by DNA sequence motifs that are present in both the viral replication origin and the chromosomal preintegration DNA and that specify binding and nicking sites for the viral regulatory Rep protein. This finding raised the question as to whether other parvovirus regulatory proteins might direct site-specific recombination with DNA targets that contain origin sequences functionally equivalent to those described for AAV. To investigate this question, active and inactive forms of the minute virus of mice (MVM) 3' replication origin, derived from a replicative-form dimer-bridge intermediate, were propagated in an Epstein-Barr virus-based shuttle vector which replicates as an episome in a cell-cycle-dependent manner in mammalian cells. Upon MVM infection of these cells, the infecting genome integrated into episomes containing the active-origin sequence reported to be efficiently nicked by the MVM regulatory protein NS1. In contrast, MVM did not integrate into episomes containing either the inactive form of the origin sequence reported to be inefficiently nicked by NS1 or the active form from which the NS1 consensus nick site had been deleted. The structure of the cloned MVM episomal recombinants displayed several features previously described for AAV episomal and chromosomal recombinants. The findings indicate that the rules which govern AAV site-specific recombination also apply to MVM and suggest that site-specific chromosomal insertions may be achievable with different autonomous parvovirus replicator proteins which recognize binding and nicking sites on the target DNA.     

Spontaneous mutagenesis of a plant potyvirus genome after insertion of a foreign gene

The RNA genome of tobacco etch potyvirus (TEV) was engineered to express bacterial beta-glucuronidase (GUS) fused to the virus helper component proteinase (HC-Pro). It was shown previously that prolonged periods (approximately 1 month) of TEV-GUS propagation in plants resulted in the appearance of spontaneous deletion variants. Nine deletion mutants were identified by nucleotide sequence analysis of 40 cDNA clones obtained after polymerase chain reaction amplification. The mutants were missing between 1,741 and 2,074 nucleotides from TEV-GUS, including the sequences coding for most of GUS and the N-terminal region of HC-Pro. This region of HC-Pro contains determinants involved in helper component activity during aphid transmission, as well as a highly conserved series of cysteine residues. The deletion variants were shown to replicate and move systemically without the aid of a helper virus. Infectious viruses harboring the two largest HC-Pro deletions (termed TEV-2del and TEV-7del) were reconstructed by subcloning the corresponding mutated regions into full-length DNA copies of the TEV genome. Characterization of these and additional variants derived by site-directed mutagenesis demonstrated that deletion of sequences coding for the HC-Pro N-terminal domain had a negative effect on accumulation of viral RNA and coat protein. The TEV-2del variant possessed an aphid-nontransmissible phenotype that could be rescued partially by prefeeding of aphids on active HC-Pro from another potyvirus. These data suggest that the N-terminal domain of HC-Pro or its coding sequence enhances virus replication or genome expression but does not provide an activity essential for these processes. The function of this domain, as well as a proposed deletion mechanism involving nonhomologous recombination, is discussed.     

The herpes simplex virus 1 protein kinase US3 is required for protection from apoptosis induced by the virus

An earlier report showed that a disabled mutant lacking both copies of the major regulatory gene (alpha4) of herpes simplex virus 1 induced DNA degradation characteristic of apoptosis in infected cells, whereas the wild-type virus protected cells from apoptosis induced by thermal shock. More extensive analyses of the disabled mutant revealed a second mutation which disabled US3, a viral gene encoding a protein kinase known to phosphorylate serine/threonine within a specific arginine-rich consensus sequence. Analyses of cells infected with a viral mutant carrying a wild-type alpha4 gene but from which the US3 gene had been deleted showed that it induced fragmentation of cellular DNA, whereas a recombinant virus in which the deleted sequences of the US3 gene had been restored did not cause the cellular DNA to fragment. These results point to the protein kinase encoded by the US3 gene as the principal viral product required to block apoptosis.