Journal of Virology

The cytoplasmic tail of the influenza A virus M2 proton-selective ion channel has been shown to be important for virus replication. Previous analysis of M2 cytoplasmic tail truncation mutants demonstrated a defect in incorporation of viral RNA (vRNA) into virions, suggesting a role for M2 in the recruitment of M1-vRNA complexes. To further characterize the effect of the M2 cytoplasmic tail mutations on virus assembly and budding, we constructed a series of alanine substitution mutants of M2 with mutations in the cytoplasmic tail, from residues 71 to 97. Mutant proteins M2-Mut1 and M2-Mut2, with mutations of residues 71 to 73 and 74 to 76, respectively, appeared to have the greatest effect on virus-like particle and virus budding, showing a defect in M1 incorporation. Mutant viruses containing M2-Mut1 and M2-Mut2 failed to replicate in multistep growth analyses on wild-type (wt) MDCK cells and were able to form plaques only on MDCK cells stably expressing wt M2 protein. Compared to wt M2 protein, M2-Mut1 and M2-Mut2 were unable to efficiently coimmunoprecipitate with M1. Furthermore, statistical analysis of planar sheets of membrane from cells infected by virus containing M2-Mut1 revealed a reduction in M1-hemagglutinin (HA) and M2-HA clustering as well as a severe loss of clustering between M1 and M2. These results suggest an essential, direct interaction between the cytoplasmic tail of M2 and M1 that promotes the recruitment of the internal viral proteins and vRNA to the plasma membrane for efficient virus assembly to occur.

We are studying the structural proteins and molecular interactions required for formation and release of influenza virus-like particles (VLPs) from the cell surface. To investigate these events, we generated a quadruple baculovirus recombinant that simultaneously expresses in Sf9 cells the hemagglutinin (HA), neuraminidase (NA), matrix (M1), and M2 proteins of influenza virus A/Udorn/72 (H3N2). Using this quadruple recombinant, we have been able to demonstrate by double-labeling immunofluorescence that matrix protein (M1) localizes in nuclei as well as at discrete areas of the plasma membrane where HA and NA colocalize at the cell surface. Western blot analysis of cell supernatant showed that M1, HA, and NA were secreted into the culture medium. Furthermore, these proteins comigrated in similar fractions when concentrated supernatant was subjected to differential centrifugation. Electron microscopic examination (EM) of these fractions revealed influenza VLPs bearing surface projections that closely resemble those of wild-type influenza virus. Immunogold labeling and EM demonstrated that the HA and NA were present on the surface of the VLPs. We further investigated the minimal number of structural proteins necessary for VLP assembly and release using single-gene baculovirus recombinants. Expression of M1 protein alone led to the release of vesicular particles, which in gradient centrifugation analysis migrated in a similar pattern to that of the VLPs. Immunoprecipitation of M1 protein from purified M1 vesicles, VLPs, or influenza virus showed that the relative amount of M1 protein associated with M1 vesicles or VLPs was higher than that associated with virions, suggesting that particle formation and budding is a very frequent event. Finally, the HA gene within the quadruple recombinant was replaced either by a gene encoding the G protein of vesicular stomatitis virus or by a hybrid gene containing the cytoplasmic tail and transmembrane domain of the HA and the ectodomain of the G protein. Each of these constructs was able to drive the assembly and release of VLPs, although enhanced recruitment of the G glycoprotein onto the surface of the particle was observed with the recombinant carrying a G/HA chimeric gene. The described approach to assembly of wild-type and chimeric influenza VLPs may provide a valuable tool for further investigation of viral morphogenesis and genome packaging as well as for the development of novel vaccines.

The therapeutic efficacy of direct drug infusion into the brain, the target organ of transmissible spongiform encephalopathies, was assessed in transgenic mice intracerebrally infected with 263K scrapie agent. Pentosan polysulfate (PPS) gave the most dramatic prolongation of the incubation period, and amphotericin B had intermediate effects, but antimalarial drugs such as quinacrine gave no significant prolongation. Treatment with the highest dose of PPS at an early or late stage of the infection prolonged the incubation time by 2.4 or 1.7 times that of the control mice, respectively. PPS infusion decreased not only abnormal prion protein deposition but also neurodegenerative changes and infectivity. These alterations were observed within the brain hemisphere fitted with an intraventricular infusion cannula but not within the contralateral hemisphere, even at the terminal disease stage long after the infusion had ended. Therapeutic effects of PPS were also demonstrated in mice infected with either RML agent or Fukuoka-1 agent. However, at doses higher than that providing the maximal effects, intraventricular PPS infusion caused adverse effects such as hematoma formation in the experimental animals. These findings indicate that intraventricular PPS infusion might be useful for the treatment of transmissible spongiform encephalopathies in humans, providing that the therapeutic dosage is carefully evaluated.

MuMig (murine monokine induced by gamma interferon) and Crg-2 (cytokine responsive gene 2) are two murine chemokines of the CXC family that are induced by the interferons (IFNs): MuMig specifically by IFN-γ and Crg-2 by IFN-α, IFN-β, and IFN-γ. To investigate the biological roles of these chemokines, recombinant vaccinia viruses (rVVs) encoding either MuMig or Crg-2 were constructed. In vitro, the chemokine-encoding rVVs replicated to similar levels to the control virus. Athymic nude mice inoculated with 10⁵PFU or less of VV-HA-Mig or VV-HA–Crg-2 resolved the infection successfully whereas mice given a similar dose of the control virus VV-HA-TK died from generalized infection. At higher doses, there was mortality in all groups but death was significantly delayed in mice infected with either chemokine-encoding rVV compared with those infected with the control virus. Virus-encoded MuMig and Crg-2 enhanced the cytolytic activity of NK cells and splenic cellularity by two- to threefold and resulted in significant increases in mononuclear cell infiltration in the livers of mice. Using specific neutralizing or depleting antibodies, we have established that the control of rVV replication in athymic nude mice, as a consequence of virus-expressed MuMig and Crg-2, requires NK cells and IFN-α, IFN-β, and IFN-γ.

The VP40 matrix protein of Ebola virus buds from cells in the form of virus-like particles (VLPs) and plays a central role in virus assembly and budding. In this study, we utilized a functional budding assay and cotransfection experiments to examine the contributions of the glycoprotein (GP), nucleoprotein (NP), and VP24 of Ebola virus in facilitating release of VP40 VLPs. We demonstrate that VP24 alone does not affect VP40 VLP release, whereas NP and GP enhance release of VP40 VLPs, individually and to a greater degree in concert. We demonstrate further the following: (i) VP40 L domains are not required for GP-mediated enhancement of budding; (ii) the membrane-bound form of GP is necessary for enhancement of VP40 VLP release; (iii) NP appears to physically interact with VP40 as judged by detection of NP in VP40-containing VLPs; and (iv) the C-terminal 50 amino acids of NP may be important for interacting with and enhancing release of VP40 VLPs. These findings provide a more complete understanding of the role of VP40 and additional Ebola virus proteins during budding.

The influenza A virus NEP (NS2) protein is an structural component of the viral particle. To investigate whether this protein has an effect on viral RNA synthesis, we examined the expression of an influenza A virus-like chloramphenicol acetyltransferase (CAT) RNA in cells synthesizing the four influenza A virus core proteins (nucleoprotein, PB1, PB2, and PA) and NEP from recombinant plasmids. Influenza A virus NEP inhibited drastically, and in a dose-dependent manner, the level of CAT expression mediated by the recombinant influenza A virus polymerase. This inhibitory effect was not observed in an analogous artificial system in which expression of a synthetic CAT RNA is mediated by the core proteins of an influenza B virus. This result ruled out the possibility that inhibition of reporter gene expression was due to a general toxic effect induced by NEP. Analysis of the virus-specific RNA species that accumulated in cells expressing the type A recombinant core proteins and NEP showed that there was an important reduction in the levels of minireplicon-derived vRNA, cRNA, and mRNA molecules. Taken together, the results obtained suggest a regulatory role for NEP during virus-specific RNA synthesis, and this finding is discussed regarding the biological implications for the virus life cycle.

Under most conditions of growth, the most abundant protein in the outer membrane of most strains of Escherichia coli is a protein designated as “protein 1” or “matrix protein”. In E. coli B, this protein has been shown to be a single polypeptide with a molecular mass of 36,500 and it may account for more than 50% of the total outer membrane protein. E. coli K-12 contains a very similar, although probably not identical, species of protein 1. Some pathogenic E. coli strains contain very little protein 1 and, in its place, make a protein designated as protein 2 which migrates faster on alkaline polyacrylamide gels containing sodium dodecyl sulfate and which gives a different spectrum of CNBr peptides. An E. coli K-12 strain which had been mated with a pathogenic strain was found to produce protein 2, and a temperate bacteriophage was isolated from this K-12 strain after induction with UV light. This phage, designated as PA-2, is similar in morphology and several other properties to phage lambda. When strains of E. coli K-12 are lysogenized by phage PA-2, they produce protein 2 and very little protein 1. Adsorption to lysogenic strains grown under conditions where they produce little protein 1 and primarily protein 2 is greatly reduced as compared to non-lysogenic strains which produce only protein 1. However, when cultures are grown under conditions of catabolite repression, protein 2 is reduced and protein 1 is increased, and lysogenic and non-lysogenic cultures grown under these conditions exhibit the same rate of adsorption. Phage PA-2 does not adsorb to E. coli B, which appears to have a slightly different protein 1 from K-12. These results suggest that protein 1 is the receptor for PA-2, and that protein 2 is made to reduce the superinfection of lysogens.

The human papillomavirus type 16 (HPV-16) E7 oncoprotein rapidly induces centrosome duplication errors in primary human cells, thereby increasing the propensity for multipolar mitoses, which can lead to chromosome missegregation and aneuploidy. We analyzed a series of HPV-16 E7 mutants and demonstrate that this biological activity of the E7 oncoprotein is mediated by sequences encompassing the core pRB binding site but is independent of its ability to inactivate the retinoblastoma tumor suppressor protein pRB and the related pocket proteins p107 and p130. In addition, interaction of E7 with the S4 subunit of the 26S proteasome and dysregulation of cdc25A transcription are also dispensable for the induction of centrosome duplication errors. Consistent with these results, expression of HPV-16 E7 induces abnormal centrosome duplication in a cell line that lacks functional pRB and in mouse embryo fibroblasts that are deficient for pRB, p107, and p130. These results demonstrate that the molecular mechanism whereby HPV-16 E7 induces centrosome duplication errors is independent of its ability to inactivate pRB, p107, and p130 or to interact with the S4 proteasome subunit.

Comparative surface feature analyses of the VP1 sequences of hepatitis A virus (HAV) and poliovirus type 1 allowed an alignment of the two sequences and an identification of probable HAV neutralization antigenic sites. A synthetic peptide containing the HAV-specific amino acid sequence of one of these sites induced anti-HAV-neutralizing antibodies. It is concluded that a structural homology exists between the two viruses, despite minimal primary sequence conservation.

AD101 and SCH-C are two chemically related small molecules that inhibit the entry of human immunodeficiency virus type 1 (HIV-1) via human CCR5. AD101 also inhibits HIV-1 entry via rhesus macaque CCR5, but SCH-C does not. Among the eight residues that differ between the human and macaque versions of the coreceptor, only one, methionine-198, accounts for the insensitivity of macaque CCR5 to inhibition by SCH-C. Thus, the macaque coreceptor engineered to contain the natural human CCR5 residue (isoleucine) at position 198 is sensitive to HIV-1 entry inhibition by SCH-C, whereas a human CCR5 mutant containing the corresponding macaque residue (methionine) is resistant. Position 198 is in CCR5 transmembrane (TM) helix 5 and is not located within the previously defined binding site for AD101 and SCH-C, which involves residues in TM helices 1, 2, 3, and 7. SCH-C binds to human CCR5 whether residue 198 is isoleucine or methionine, and it also binds to macaque CCR5. However, the binding of a conformation-dependent monoclonal antibody to human CCR5 is inhibited by SCH-C only when residue 198 is isoleucine. These observations, taken together, suggest that the antiviral effects of SCH-C and AD101 involve stabilization, or induction, of a CCR5 conformation that is not compatible with HIV-1 infection. However, SCH-C is unable to exert this effect on CCR5 conformation when residue 198 is methionine. The region of CCR5 near residue 198 has, therefore, an important influence on the conformational state of this receptor.