Mutations altering the moloney murine leukemia virus p12 Gag protein affect virion production and early events of the virus life cycle.

The p12 Gag protein of Moloney murine leukemia virus is a small polypeptide of unknown function, containing two proline-rich motifs. To determine its role in replication, we introduced a series of deletion and alanine-scanning substitution mutations throughout the p12 coding region of a proviral DNA, and characterized the phenotypes of the resulting mutant viruses. Complete deletion of p12 and mutations affecting the PPPY motif caused substantial reduction in the yield of virions and a modest reduction in Gag processing. Proteolytic cleavage of the R-peptide from the cytoplasmic tail of the envelope protein TM was abolished in these mutants, suggesting that the PPPY motif is crucial for the viral protease to access the TM tail. The resulting virions were non-infectious, and unable to initiate DNA synthesis in infected cells. Mutants with alterations in both the N- and C-terminal portions of p12 exhibited a distinct phenotype. The production of virions and processing of Gag, Pol and Env precursors were normal. The viruses were able to direct synthesis of linear viral DNA, but there was almost no detectable circular DNAs or LTR-LTR junction. These data suggest that p12 plays a critical role in the early events of the virus life cycle.     

The integrated genome of murine leukemia virus

The Southern gel filter transfer technique has been used to characterize the integrated genome of Moloney murine leukemia virus (M-MuLV) and the genomes of the endogenous viruses of the mouse. Study of 10 clones of rat cell independently infected by M-MuLV indicates a minimum of 15 integration sites into which the M-MuLV provirus can be inserted. No common integration site is observed among these clones. Clones productively infected by M-MuLV acquire multiple proviruses, whereas infected cells unable to produce virus contain only one M-MuLV provirus. Once established, the integrated genomes are stable for at least two years after initial infection.The use of M-MuLV probe allows detection of a spectrum of Eco RI-cleaved mouse DNA fragments containing endogenous MuLV genomes. DNAs of different inbred laboratory mouse strains yield similar patterns of provirus with each strain showing minor characteristic differences. In some instances, mouse cells infected by M-MuLV reveal additional proviruses beyond those seen in the uninfected cell. DNAs from three different M-MuLV-induced thymomas indicate, as in rat cells, multiple possible integration sites.     

Phosphorylated serine residues and an arginine-rich domain of the moloney murine leukemia virus p12 protein are required for early events of viral infection

Mutational analyses of the p12 Gag phosphoprotein of Moloney murine leukemia virus have demonstrated its participation in both virus assembly and the early stages of infection. The molecular mechanisms by which p12 functions in these events are still poorly understood. We performed studies to examine the significance of p12 phosphorylation in the viral life cycle. Alanine substitutions were introduced at the potential phosphorylation sites in p12, and the resulting mutants were tested for replication. Mutant viruses with changes at S61 and S78 were severely impaired, whereas the other mutant viruses were viable. S61 was shown to be required for normal levels of phosphorylation of p12 in vivo. These defective mutant viruses showed no apparent alteration to Gag protein processing or reduction in the yield of virions after transient transfection, but the mutants failed to form circular viral DNAs in acutely infected cells. Sequence analysis of revertant clones derived from S(61,65)A mutant virus revealed two classes: one group with a single mutation at a residue adjacent to S61 and another group with mutations introducing new positive charges surrounding S61. In vivo [32P]orthophosphate labeling indicated that the rescue of the S(61,65)A mutant virus did not result in a significant increase in the phosphorylation level of p12. Alanine substitutions of an arginine-rich stretch near S61 (at R-66, -68, -70, and -71) resulted in the same phenotype as the S(61,65)A mutant virus. The restored function of S(61,65)A mutant virus by second or third site mutations may result from a structural change or the addition of positively charged residues in the arginine-rich region.     

Characterization of avian myeloblastosis-associated virus DNA intermediates.

The major species of unintegrated linear viral DNA identified in chicken embryonic fibroblasts infected with either the avian myeloblastosis-associated viruses (MAV-1, MAV-2) or the standard avian myeloblastosis virus complex (AMV-S) has a mass of 5.3 X 10(6) daltons. An additional minor DNA component observed only in AMV-S-infected cells has a mass of 4.9 X 10(6) daltons. The unintegrated linear viral DNAs and integrated proviruses of MAV-1 and MAV-2 have been analyzed by digestion with the restriction endonucleases EcoRI and HindIII. MAV-2 lacks a HindIII site present in MAV-1. These fragments have been compared to those generated by EcoRI and HindIII digestion of linear viral DNAs of AMV-S. Restriction enzyme digestion of AMV-S viral DNA produced unique fragments not found with either MAV-1 or MAV-2 viral DNAs. The major viral component present in AMV-S stocks has the HindIII restriction pattern of MAV-1. Restriction enzyme analysis of the 5.3 X 10(6)-dalton unintegrated MAV viral DNAs and their integrated proviruses suggests that the DNAs have a direct terminal redundancy of approximately 0.3 megadaltons and integrate colinearly with respect to the unintegrated linear DNA.     

Multiple integrase functions are required to form the native structure of the human immunodeficiency virus type I intasome.

Mu-mediated polymerase chain reaction footprinting was used to investigate the protein-DNA structure of human immunodeficiency virus type I (HIV-I) preintegration complexes. Preintegration complexes were partially purified from cells after using an established coculture infection technique as well as a novel technique using cell-free supernatant from transfected cells as the source of virus. Footprinting revealed that bound proteins protected the terminal 200-250 base pairs of each viral end from nuclease attack. Bound proteins also caused strong transpositional enhancements near each end of HIV-I. In contrast, regions of viral DNA internal to the ends did not show evidence of strong protein binding. The end regions of preintegrative HIV-I apparently form a unique nucleoprotein structure, which we term the intasome to distinguish it from the greater preintegration complex. Our novel system also allowed us to analyze the structure and function of preintegration complexes isolated from cells infected with integrase mutant viruses. Complexes were derived from viruses defective for either integrase catalysis, integrase binding to the viral DNA substrate, or an unknown function in the carboxyl-terminal domain of the integrase protein. None of these mutant complexes supported detectable integration activity. Despite the presence of the mutant integrase proteins in purified samples, none of these nucleoprotein complexes displayed the native intasome structure detected in wild-type preintegration complexes. We conclude that multiple integrase functions are required to form the native structure of the HIV-I intasome in infected cells.     

Roles for the E4 orf6, orf3, and E1B 55-Kilodalton Proteins in Cell Cycle-Independent Adenovirus Replication

Adenoviruses bearing lesions in the E1B 55-kDa protein (E1B 55-kDa) gene are restricted by the cell cycle such that mutant virus growth is most impaired in cells infected during G(1) and least restricted in cells infected during S phase (F. D. Goodrum and D. A. Ornelles, J. Virol. 71:548-561, 1997). A similar defect is reported here for E4 orf6-mutant viruses. An E4 orf3-mutant virus was not restricted for growth by the cell cycle. However, orf3 was required for enhanced growth of an E4 orf6-mutant virus in cells infected during S phase. The cell cycle restriction may be linked to virus-mediated mRNA transport because both E1B 55-kDa- and E4 orf6-mutant viruses are defective at regulating mRNA transport at late times of infection. Accordingly, the cytoplasmic-to-nuclear ratio of late viral mRNA was reduced in G(1) cells infected with the mutant viruses compared to that in G(1) cells infected with the wild-type virus. By contrast, this ratio was equivalent among cells infected during S phase with the wild-type or mutant viruses. Furthermore, cells infected during S phase with the E1B 55-kDa- or E4 orf6-mutant viruses synthesized more late viral protein than did cells infected during G(1). However, the total amount of cytoplasmic late viral mRNA was greater in cells infected during G(1) than in cells infected during S phase with either the wild-type or mutant viruses, indicating that enhanced transport of viral mRNA in cells infected during S phase cannot account for the difference in yields in cells infected during S phase and in cells infected during G(1). Thus, additional factors affect the cell cycle restriction. These results indicate that the E4 orf6 and orf3 proteins, in addition to the E1B 55-kDa protein, may cooperate to promote cell cycle-independent adenovirus growth.     

Infectivity of Moloney murine leukemia virus defective in late assembly events is restored by late assembly domains of other retroviruses

The p12 region of the Moloney murine leukemia virus (M-MuLV) Gag protein contains a PPPY motif important for efficient virion assembly and release. To probe the function of the PPPY motif, a series of insertions of homologous and heterologous motifs from other retroviruses were introduced at various positions in a mutant gag gene lacking the PPPY motif. The assembly defects of the PPPY deletion mutant could be rescued by insertion of a wild-type PPPY motif and flanking sequences at several ectopic positions in the Gag protein. The late assembly domain (L-domain) of Rous sarcoma virus (RSV) or human immunodeficiency virus type 1 (HIV-1) could also fully or partially restore M-MuLV assembly when introduced into matrix, p12, or nucleocapsid domains of the mutant M-MuLV Gag protein lacking the PPPY motif. Strikingly, mutant viruses carrying the RSV or the HIV-1 L-domain at the original location of the deleted PPPY motif were replication competent in rodent cells. These data suggest that the PPPY motif of M-MuLV acts in a partially position-independent manner and is functionally interchangeable with L-domains of other retroviruses. Electron microscopy studies revealed that deletion of the entire p12 region resulted in the formation of tube-like rather than spherical particles. Remarkably, the PPPY deletion mutant formed chain structures composed of multiple viral particles linked on the cell surface. Many of the mutants with heterologous L-domains released virions with wild-type morphology.     

Status of Marek's disease virus in established lymphoma cell lines: herpesvirus integration is common.

Six cell lines derived from Marek's disease lymphomas of chickens and turkeys were investigated for the status of Marek's disease virus (MDV) DNA. In the transformed T- and B-cell lines, viral DNA could be detected by conventional Southern blot hybridization, by Gardella gel electrophoresis, and by in situ hybridization of metaphase and interphase chromosomes. Integration of viral DNA into the host cell chromosome was observed in all cell lines. Two to 12 integration sites of viral DNA could be detected in metaphase chromosome spreads. The integration sites were characteristic for the individual cell lines and were preferentially located at the telomers of large- and mid-sized chromosomes or on minichromosomes. In four of six cell lines, a minor population of latently infected cells supported the lytic cycle of MDV, giving rise to linear virion DNAs. In one of these cell lines, a third species of MDV DNA could be detected with properties reminiscent of covalently closed circular DNA. The finding that MDV integrates regularly into the genomes of latently infected cells is crucial to understanding the molecular biology of herpesvirus-induced tumors in the natural host.     

The early region 1B 55-kilodalton oncoprotein of adenovirus relieves growth restrictions imposed on viral replication by the cell cycle.

The E1B 55-kDa oncoprotein of adenovirus enables the virus to overcome restrictions imposed on viral replication by the cell cycle. Approximately 20% of HeLa cells infected with an E1B 55-kDa mutant adenovirus produced virus when evaluated by electron microscopy or by assays for infectious centers. By contrast, all HeLa cells infected with a wild-type adenovirus produced virus. The yield of E1B mutant virus from randomly cycling HeLa cells correlated with the fraction of cells in S phase at the time of infection. In synchronously growing HeLa cells, approximately 75% of the cells infected during S phase with the E1B mutant virus produced virus, whereas only 10% of the cells infected during G1 produced virus. The yield of E1B mutant virus from HeLa cells infected during S phase was sevenfold greater than that of cells infected during G1 and threefold greater than that of cells infected during asynchronous growth. Cells infected during S phase with the E1B mutant virus exhibited severe cytopathic effects, whereas cells infected with the E1B mutant virus during G1 exhibited a mild cytopathic effect. Viral DNA synthesis appeared independent of the cell cycle because equivalent amounts of viral DNA were synthesized in cells infected with either wild-type or E1B mutant virus. The inability of the E1B mutant virus to replicate was not mediated by the status of p53. These results define a novel property of the large tumor antigen of adenovirus in relieving growth restrictions imposed on viral replication by the cell cycle.     

Measurement of the Sequence Complexity of Cloned Moloney Murine Leukemia Virus 60 to 70S RNA: Evidence for a Haploid Genome

The sequence complexity of the 60-70S RNA complex from Moloney murine leukemia virus (M-MuLV) was determined by measuring the annealing rate of radioactively labeled virus-specific DNA with M-MuLV 60-70S RNA in conditions of vast RNA excess. The M-MuLV RNA annealing rate, characterized by the quantity C(r)t((1/2)), was compared with the C(r)t((1/2)) values for annealing of poliovirus 35S RNA (2.6 x 10(6) molecular weight) with poliovirus-specific DNA and Sindbis virus 42S RNA (4.3 x 10(6) molecular weight) with Sindbis-specific DNA. M-MuLV-specific DNA was prepared in vitro by the endogenous DNA polymerase reaction of M-MuLV virions, and poliovirus and Sindbis virus DNAs were prepared by incubation of viral RNA and DNA polymerase purified from avian myeloblastosis virus and an oligo deoxynucleotide primer. The poliovirus and Sindbis virus DNAs were sedimented through alkaline sucrose gradients, and those portions of the DNA with sizes similar to the M-MuLV DNA were selected out for the annealing measurements. M-MuLV was cloned on NIH-3T3 cells because it appeared possible that the standard source of M-MuLV for these experiments was a mixture of viruses. The annealing measurements indicated a sequence complexity of approximately 9 x 10(6) daltons for the cloned M-MuLV 60-70S RNA when standardized to poliovirus and Sindbis virus RNAs. This value supports the hypothesis that each of the 35S RNA subunits of M-MuLV 60-70S RNA has a different base sequence.     

Roles of the auxiliary genes and AP-1 binding site in the long terminal repeat of feline immunodeficiency virus in the early stage of infection in cats.

To examine the roles of auxiliary genes and the AP-1 binding site in the long terminal repeat of feline immunodeficiency virus (FIV) in vivo, three mutant viruses, which are defective in the vif gene ([delta]vif), ORF-A gene (deltaORF-A), and AP-1 binding site (deltaAP-1), and wild-type virus as a positive control were separately inoculated into three specific-pathogen-free cats. These cats were assessed by measuring the number of proviral DNA copies in peripheral blood mononuclear cells (PBMCs), the CD4/CD8 ratio and antibody responses to FIV for 16 weeks and then examining histological changes at necropsy. Although viral DNAs were detected in PBMCs from all 12 cats to various degrees until 16 weeks postinoculation, no virus was recovered from PBMCs of cats infected with (delta)vif virus during the observation period. However, a very weak antibody response was induced in one cat infected with the (delta)vif virus. In contrast, despite the successful recovery of virus from both groups of cats infected with deltaORF-A and deltaAP-1 virus, antibody responses and decrease in the CD4/CD8 ratio in the groups were milder than those in cats infected with wild-type virus. Furthermore, the numbers of proviral DNA copies in PBMCs from the two groups were not able to reach the level in cats infected with wild-type virus during the observation period. From these results, we conclude that these mutant viruses are still infectious for cats but failed in efficient viral replication and suggest that these auxiliary genes and enhancer element are important or essential to full viral replication kinetics and presumably to full pathogenicity during the early stage of infection in vivo.     

Different modes of human papillomavirus DNA replication during maintenance

Human papillomavirus (HPV) begins its life cycle by infecting the basal cells of the epithelium. Within these proliferating cells, the viral genomes are replicated, maintained, and passed on to the daughter cells. Using HPV episome-containing cell lines that were derived from naturally infected cervical tissues, we investigated the mode by which the viral DNAs replicate in these cells. We observed that, whereas HPV16 DNA replicated in an ordered once-per-S-phase manner in W12 cells, HPV31 DNA replicated via a random-choice mechanism in CIN612 cells. However, when HPV16 and HPV31 DNAs were separately introduced into an alternate keratinocyte cell line NIKS, they both replicated randomly. This indicates that HPV DNA is inherently capable of replicating by either random-choice or once-per-S-phase mechanisms and that the mode of HPV DNA replication is dependent on the cells that harbor the viral episome. High expression of the viral replication protein E1 in W12 cells converted HPV16 DNA replication to random-choice replication and, as such, it appears that the mode of HPV DNA replication in proliferating cells is dependent on the presence or the increased level of this protein in the host cell. The implications of these observations on maintenance, latency, and persistence are discussed.     

The Gag cleavage product, p12, is a functional constituent of the murine leukemia virus pre-integration complex

The p12 protein is a cleavage product of the Gag precursor of the murine leukemia virus (MLV). Specific mutations in p12 have been described that affect early stages of infection, rendering the virus replication-defective. Such mutants showed normal generation of genomic DNA but no formation of circular forms, which are markers of nuclear entry by the viral DNA. This suggested that p12 may function in early stages of infection but the precise mechanism of p12 action is not known. To address the function and follow the intracellular localization of the wt p12 protein, we generated tagged p12 proteins in the context of a replication-competent virus, which allowed for the detection of p12 at early stages of infection by immunofluorescence. p12 was found to be distributed to discrete puncta, indicative of macromolecular complexes. These complexes were localized to the cytoplasm early after infection, and thereafter accumulated adjacent to mitotic chromosomes. This chromosomal accumulation was impaired for p12 proteins with a mutation that rendered the virus integration-defective. Immunofluorescence demonstrated that intracellular p12 complexes co-localized with capsid, a known constituent of the MLV pre-integration complex (PIC), and immunofluorescence combined with fluorescent in situ hybridization (FISH) revealed co-localization of the p12 proteins with the incoming reverse transcribed viral DNA. Interactions of p12 with the capsid and with the viral DNA were also demonstrated by co-immunoprecipitation. These results imply that p12 proteins are components of the MLV PIC. Furthermore, a large excess of wt PICs did not rescue the defect in integration of PICs derived from mutant p12 particles, demonstrating that p12 exerts its function as part of this complex. Altogether, these results imply that p12 proteins are constituent of the MLV PIC and function in directing the PIC from the cytoplasm towards integration.     

Sites of integration of infectious DNA of avian reticuloendotheliosis viruses in different avian cellular DNAs.

The pattern of integration for the infectious DNA of two avian reticuloendotheliosis viruses whose DNA is not inactivated by digestion with the restriction endonuclease, Eco RI was determined. High molecular weight DNA from infected chicken, turkey and pheasant cells was digested with Eco RI, electrophoresed through agarose gels and assayed for infectivity. The same patterns of integration of infectious viral DNA were found for these species of avian cells infected at high or low multiplicities with two reticuloendotheliosis viruses. There were multiple sites of integration in acutely infected cells with concomitant cell death. There was a single site of integration in chronically infected cells with no cell death. There were more integrated infectious viral DNA molecules per cell in acutely infected cells than in chronically infected cells. These results are consistent with the hypotheses that the cell death in the acute phase of infection is a result of the integration of the infectious viral DNA at multiple sites, and that only those cells survive that have the infectious viral DNA integrated exclusively at the single site.     

Human cytomegalovirus disrupts both ataxia telangiectasia mutated protein (ATM)- and ATM-Rad3-related kinase-mediated DNA damage responses during lytic infection

Many viruses (herpes simplex virus type 1, polyomavirus, and human immunodeficiency virus type 1) require the activation of ataxia telangiectasia mutated protein (ATM) and/or Mre11 for a fully permissive infection. However, the longer life cycle of human cytomegalovirus (HCMV) may require more specific interactions with the DNA repair machinery to maximize viral replication. A prototypical damage response to the double-stranded ends of the incoming linear viral DNA was not observed in fibroblasts at early times postinfection (p.i.). Apparently, a constant low level of phosphorylated ATM was enough to phosphorylate its downstream targets, p53 and Nbs1. p53 was the only cellular protein observed to relocate at early times, forming foci in infected cell nuclei between 3.5 and 5.5 h p.i. Approximately half of these foci localized with input viral DNA, and all localized with viral UL112/113 prereplication site foci. No other DNA repair proteins localized with the virus or prereplication foci in the first 24 h p.i. When viral replication began in earnest, between 24 and 48 h p.i., there were large increases in steady-state levels and phosphorylation of many proteins involved in the damage response, presumably triggered by ATM-Rad3-related kinase activation. However, a sieving process occurred in which only certain proteins were specifically sequestered into viral replication centers and others were particularly excluded. In contrast to other viruses, activation of a damage response is neither necessary nor detrimental to infection, as neither ATM nor Mre11 was required for full virus replication and production. Thus, by preventing simultaneous relocalization of all the necessary repair components to the replication centers, HCMV subverts full activation and completion of both double-stranded break and S-phase checkpoints that should arrest all replication within the cell and likely lead to apoptosis.     

Integrated Moloney murine leukemia virus DNA studied by using complementary DNA which does not recognize endogenous related sequences

By preannealing a radioactive, representative Moloney murine leukemia virus (M-MuLV) cDNA with large excesses of AKR 70S viral RNA, an M-MuLV-specific cDNA has been prepared. When hybridized to restriction enzyme fragments of M-MuLV-infected mouse cell DNA, the preannealed probe recognizes integrated M-MuLV DNA and does not recognize endogenous related DNA sequences found in uninfected mouse cells. The viral DNA sequences recognized by the preannealed probe are spread throughout the viral genome, although some sequences are recognized less efficiently. By using this preannealed probe, multiple integrations of M-MuLV DNA have been detected in infected fibroblasts and in an M-MuLV-induced tumor. Integrated viral DNA fragments smaller than the complete viral genome have also been detected. By using this preannealed probe to examine a mass-infected culture of mouse fibroblasts, no evidence for a strongly preferred site for M-MuLV integration could be found.     

n-Butyrate, a cell cycle blocker, inhibits the replication of polyomaviruses and papillomaviruses but not that of adenoviruses and herpesviruses.

Small DNA viruses are dependent on the interaction of early proteins (such as large T antigen) with host p53 and Rb to bring about the G1-to-S cell cycle transition. The large DNA viruses are less dependent on host regulatory genes since additional early viral proteins (such as viral DNA polymerase, DNA metabolic enzymes, and other replication proteins) are involved in DNA synthesis. A highly conserved domain of large T antigen (similar to the p53-binding region) exclusively identifies papovavirus, parvovirus, and papillomaviruses from all other larger DNA viruses and implies a conserved interaction with host regulatory genes. In this report, we show that 3 to 6 mM butyrate, a general cell cycle blocker implicated in inhibition of the G1-to-S transition, inhibits DNA replication of polyomavirus and human papillomavirus type 11 but not the replication of larger DNA viruses such as adenovirus types 2 and 5, herpes simplex virus type 1, Epstein-Barr virus, and cytomegalovirus, which all bypass the butyrate-mediated cell cycle block. This butyrate effect on polyomavirus replication is not cell type specific, nor does it depend on the p53 or Rb gene, as inhibition was seen in fibroblasts with intact or homozygous deleted p53 or Rb, 3T6 cells, keratinocytes, C2C12 myoblasts, and 3T3-L1 adipocytes. In addition, butyrate did not inhibit expression of polyomavirus T antigen. The antiviral effect of butyrate involves a form of imprinted state, since pretreatment of cells with 3 mM butyrate inhibits human papillomavirus type 11 DNA replication for at least 96 h after its removal. Butyrate, therefore, serves as a molecular tool in dissecting the life cycle of smaller DNA viruses from that of the larger DNA viruses in relation to the cell cycle.