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.     

N-terminal cleavage fragment of glycosylated Gag is incorporated into murine oncornavirus particles

Glycosylated Gag (Glycogag) is a transmembrane protein encoded by murine and feline oncornaviruses. While the protein is dispensible for virus replication, Glycogag-null mutants of a neurovirulent murine oncornavirus are slow to spread in vivo and exhibit a loss of pathogenicity. The function of this protein in the virus life cycle, however, is not understood. Glycogag is expressed at the plasma membrane of infected cells but has not been detected in virions. In the present study we have reexamined this issue and have found an N-terminal cleavage fragment of Glycogag which was pelleted by high-speed centrifugation and sedimented in sucrose density gradients at the same bouyant density as virus particles. Its association with virions was confirmed by velocity sedimentation through iodixanol, which effectively separated membrane microvesicles from virus particles. Furthermore, the apparent molecular weight of the virion-associated protein was different from that of the protein extracted from the plasma membrane, suggesting some level of specificity or selectivity of incorporation.     

Gene product of Moloney murine leukemia virus required for proviral integration is a DNA-binding protein

The 3'-terminal portion of the retroviral pol gene encodes a function required for the formation of the integrated provirus soon after infection of sensitive cells. To permit the isolation of large quantities of the gene product, we expressed various portions of the pol gene of Moloney murine leukemia virus (M-MuLV) as trpE fusion proteins in Escherichia coli. The proteins were found to exhibit strong DNA-binding activity after extraction and renaturation by two different procedures. In the first method, proteins separated by polyacrylamide gel electrophoresis were blotted to nitrocellulose and assayed when bound to the support. The second procedure involved the isolation of proteins in an insoluble fraction, solubilization with guanidine, and renaturation. The characteristics of the binding activity are described and compared with those of authentic viral protein.     

Incorporation of human immunodeficiency virus type 1 Gag proteins into murine leukemia virus virions.

The retroviral Gag polyprotein is necessary and sufficient for assembly and budding of viral particles. However, the exact inter- and intramolecular interactions of the Gag polyproteins during this process are not known. To locate functional domains within Gag, we generated chimeric proviruses between human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MuLV). In these chimeric proviruses, the matrix or capsid proteins of MuLV were precisely replaced with the matrix or capsid proteins of HIV-1. Although the chimeric proviruses were unable to efficiently assemble into mature viral particles by themselves, coexpression of wild-type MuLV Gag rescued the HIV proteins into virions. The specificity of the rescue of HIV proteins into MuLV virions shows that specific interactions involving homologous matrix or capsid regions of Gag are necessary for retroviral particle formation.     

Characterization of Moloney murine leukemia virus p12 mutants blocked during early events of infection

Mutations affecting either the N- or C-terminal regions of the Gag protein p12 block replication of Moloney murine leukemia virus (M-MuLV). Viruses carrying mutations in this portion of gag can mediate the assembly and release of virions but are unable to successfully carry out the early phase of the M-MuLV life cycle. Wild-type and mutant viruses were found to synthesize similar levels of linear viral DNA in both cytoplasmic and nuclear fractions, and there were no significant differences in either the density or sedimentation of the viral protein-nucleic acid complexes. Analysis of the termini of the linear viral DNAs showed that the 3' ends of the mutant viral DNA were processed normally by the integrase. Further, the preintegration complexes extracted from the cytoplasm of cells infected with the mutant viruses were competent for integration into target DNA in vitro. Nevertheless, no circular viral DNAs were detected in cells infected by the mutants, and functional proviruses were not formed. These results suggest that p12 has an unexpected role in the early phase of the life cycle and is needed after viral DNA synthesis to deliver the incoming DNA to the correct location and in the appropriate state to permit either circularization or integration of the viral DNA in vivo.     

The bovine leukemia virus p34 is a transactivator protein.

Recombinant Moloney murine retroviruses containing the BLV post-envelope long open reading frame were constructed and transfected into the psi 2 packaging cell line. They were shown to encode and to express a 34-kd protein able to transactivate the BLV long terminal repeat-directed gene expression in the respective transfected cells. These data demonstrate that the BLV X-LOR gene encodes a p34 transactivator product. Furthermore, the different cell lines produced infectious recombinant retroviruses capable of transferring X-LOR genes into recipient cells. The availability of the BLV transactivator protein should allow us to understand the role of the transactivator protein in BLV-induced leukemogenesis.     

Elastase is a constituent product of T cells

Proteases produced by immune cells have been found to be important components of the immune response to antigen. A protease previously unrecognized as a specific T cell product has been identified which has the gene sequence, serologic crossreactivity, and enzymatic specificity of elastase. T cell elastase, found in combination with the natural elastase inhibitor alpha 1-antitrypsin (alpha 1-protease inhibitor, alpha 1-PI), is produced by both CD4+ and CD8+ T lymphocytes, and is found both in a membrane-bound and in a soluble form in murine T cell lines.     

Gag influences transformation by Abelson murine leukemia virus and suppresses nuclear localization of the v-Abl protein

Like the v-Onc proteins encoded by many transforming retroviruses, the v-Abl protein is expressed as a Gag-Onc fusion. Although the Gag-derived myristoylation signal targets the v-Abl protein to the plasma membrane, the protein contains the entire MA and p12 sequences and a small number of CA-derived residues. To understand the role of Gag sequences in transformation, mutants lacking portions of these sequences were examined for the effects of these deletions on v-Abl function and localization. Deletion of the N-terminal third of p12 or all of p12 enhanced the transformation of both pre-B cells and NIH 3T3 cells. In contrast, deletions in MA or a deletion removing all of Gag except the first 34 amino acids important for myristoylation highly compromised the ability to transform either cell type. Although all of the mutant proteins retained kinase activity, those defective in transformation were reduced in their ability to activate Erk, suggesting a role for Gag sequences in v-Abl signaling. Immunofluorescence analysis revealed that a v-Abl protein retaining only the first 34 amino acids of Gag localized to the nucleus. These data indicate that Gag sequences are important for normal v-Abl signaling and that they suppress nuclear localization of the molecule.     

Further studies on the glycosylated gag gene products of Rauscher murine leukemia virus: identification of an N-terminal 45,000-dalton cleavage product

A glycosylated 45,000-Mr protein containing Rauscher murine leukemia virus p15 and p12 antigenic sites and tryptic peptides was identified in Rauscher murine leukemia virus-infected cells. This glycoprotein, termed gP45gag, was also shown to contain a single tryptic peptide also present in gPr80gag and its unglycosylated apoprotein precursor Pr75gag, but lacking in Pr65gag or Pr40gag. The presence of this peptide only in viral precursor proteins containing the so-called leader (L) sequence strongly suggests that gPr45gag is an N-terminal fragment of larger glycosylated gag polyproteins, composed of L sequences in addition to p15 and p12. The kinetics of appearance of radiolabeled gPr45gag and its disappearance during chase-incubation is suggestive of a precursor-like role for this intermediate gene product. An observed 27,000-Mr glycosylated polypeptide, termed gP27gag and containing p15 but not p12, p30, or p10 antigenic determinants, is a candidate cleavage product derived from gPr45gag. These observations suggest that gPr45gag and its putative cleavage product gP27gag represent an authentic pathway for intracellular processing of glycosylated core proteins.     

Endonucleolytic cleavage of murine leukemia virus 35S RNA by microsome-associated nuclease.

Moloney murine leukemia virus 35S RNA (molecular weight 3 to 3.4 × 10⁶) is cleaved by nuclease activity present in microsomal fractions from MLV infected or uninfected mouse embryo cells to two RNA species of approximate molecular weights 1.8 × 10⁶ and 1.5 × 10⁶. Microsomal fractions from MLV infected and uninfected cells also contained nucleolytic activity that solubilized [³H]poly(A)·poly(U) but not [³H]poly(C) or [³H]poly(U); the cleavage of poly(A)·poly(U) was inhibited by ethidium bromide. The cleavage of MLV RNA was also inhibited by ethidium bromide, suggesting double stranded regions in 35S RNA as the site of cleavage.     

In vitro proteolytic cleavage of Gazdar murine sarcoma virus p65gag.

Moloney murine leukemia virus, disrupted in concentrations of 0.1 to 0.5% Nonidet P-40, catalyzed the cleavage of p65, the gag gene polyprotein of the Gazdar strain of murine sarcoma virus, into polypeptides with sizes and antigenic determinants of murine leukemia virus-specified p30, p15, pp12, and p10. Cleavage performed in the presence of 0.15% Nonidet P-40 in water yielded polypeptides of approximately 40,000 (P40) and 25,000 (P25) Mr. In vitro cleavage performed in a buffered solution containing dithiothreitol in addition to 0.1% Nonidet P-40 allowed the efficient processing of P40 to p30 and a band migrating with p10. Immunoprecipitation with monospecific sera indicated that P40 contained p30 and p10, whereas P25 contained p15 and pp12 determinants. P40 and P25 are similar in size and antigenic properties to Pr40gag and Pr25gag observed in infected cells (Naso et al, J. Virol. 32:187-198, 1979).     

R-Peptide cleavage potentiates fusion-controlling isomerization of the intersubunit disulfide in Moloney murine leukemia virus Env

Fusion of the membrane of the Moloney murine leukemia virus (Mo-MLV) Env protein is facilitated by cleavage of the R peptide from the cytoplasmic tail of its TM subunit, but the mechanism for this effect has remained obscure. The fusion is also controlled by the isomerization of the intersubunit disulfide of the Env SU-TM complex. In the present study, we used several R-peptide-cleavage-inhibited virus mutants to show that the R peptide suppresses the isomerization reaction in both in vitro and in vivo assays. Thus, the R peptide affects early steps in the activation pathway of murine leukemia virus Env.     

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.     

NMR study of xenotropic murine leukemia virus-related virus protease in a complex with amprenavir

Liver steatosis was once believed to be a benign condition, with rare progression to chronic liver disease. Thus, in both clinical and experimental practice, it is fundamental to have a reliable and objective method for its precise quantification. An image analysis algorithm was developed and validated for automatically and rapidly quantifying hepatic fat microvesicles. The image processing algorithms automatically segmented interstitial steatosis areas and analyzed the threshold region. Automatic quantifications did not significantly differ from manual evaluations of means of the same areas. Comparison of our image analysis quantifications with staging of histologic evaluations of liver steatosis presented significant correlations that are based on the distribution patterns and on the area quantity of steatosis, respectively. The use of algorithms for analysis and image processing is a sensitive, precise, objective and reproducible method of quantifying hepatic fat microvesicles, which complements semi-quantitative histologic evaluation systems.     

The PPPY motif of human T-cell leukemia virus type 1 Gag protein is required early in the budding process

Domains required late in the virus budding process (L domains) have been identified in the Gag proteins of a number of retroviruses. Here we show that the human T-cell leukemia virus type 1 candidate L domain motif PPPY is indeed required for virus production. Strikingly, however, mutation of this motif arrested virus particles at an earlier stage in the budding process than was seen for mutation of the L domain motifs thus far described for retroviruses. In view of the exchangeability of such domains, we propose that the retrovirus budding process may involve a continuum from bud formation to membrane fission.     

The tobacco mosaic virus 126-kilodalton protein, a constituent of the virus replication complex, alone or within the complex aligns with and traffics along microfilaments

Virus-induced cytoplasmic inclusion bodies (referred to as virus replication complexes [VRCs]) consisting of virus and host components are observed in plant cells infected with tobacco mosaic virus, but the components that modulate their form and function are not fully understood. Here, we show that the tobacco mosaic virus 126-kD protein fused with green fluorescent protein formed cytoplasmic bodies (126-bodies) in the absence of other viral components. Using mutant 126-kD:green fluorescent fusion proteins and viral constructs expressing the corresponding mutant 126-kD proteins, it was determined that the size of the 126-bodies and the corresponding VRCs changed in synchrony for each 126-kD protein mutation tested. Through colabeling experiments, we observed the coalignment and intracellular trafficking of 126-bodies and, regardless of size, VRCs, along microfilaments (MFs). Disruption of MFs with MF-depolymerizing agents or through virus-induced gene silencing compromised the intracellular trafficking of the 126-bodies and VRCs and virus cell-to-cell movement, but did not decrease virus accumulation to levels that would affect virus movement or prevent VRC formation. Our results indicate that (1) the 126-kD protein modulates VRC size and traffics along MFs in cells; (2) VRCs traffic along MFs in cells, possibly through an interaction with the 126-kD protein, and the negative effect of MF antagonists on 126-body and VRC intracellular movement and virus cell-to-cell movement correlates with the disruption of this association; and (3) virus movement was not correlated with VRC size.     

Leader protein of foot-and-mouth disease virus is required for cleavage of the p220 component of the cap-binding protein complex.

Suppression of host protein synthesis in cells infected by poliovirus and certain other picornaviruses involves inactivation of the cap-binding protein complex. Inactivation of this complex has been correlated with the proteolytic cleavage of p220, a component of the cap-binding protein complex. Since picornaviral RNA is not capped, it continues to be translated as the cap-binding protein complex is inactivated. The cleavage of p220 can be induced to occur in vitro, catalyzed by extracts from infected cells or by reticulocyte lysates translating viral RNA. Expression of polioviral protease 2A is sufficient to induce p220 cleavage, and the presence in 2A of an 18-amino-acid sequence representing a putative cysteine protease active site correlates with the ability of different picornaviruses to induce p220 cleavage. Foot-and-mouth disease virus (FMDV) infection induces complete cleavage of p220, yet the FMDV genome codes for a 2A protein of only 16 amino acids, which does not include the putative cysteine protease active site. Using cDNA plasmids encoding various regions of the FMDV genome, we have determined that the leader protein is required to initiate p220 cleavage. This is the first report of a function for the leader protein, other than that of autocatalytic cleavage from the FMDV polyprotein.