Michael Bruns

Lymphocytic choriomeningitis virus. VI. Isolation of a glycoprotein mediating neutralization

A structural glycoprotein of lymphocytic choriomeningitis virus was obtained in pure form by immunoaffinity chromatography using a monoclonal antibody with high neutralizing activity. It blocked neutralization of viral infectivity by antibody and in polyacrylamide gel electrophoresis it migrated with an apparent molecular weight of 44 X 10(3). We conclude that the isolated material is identical with the previously described gp44 (GP-1).

Host cell-dependent homologous interference in lymphocytic choriomeningitis virus infection

The generation of virus progeny as well as transcription, translation, and replication of the viral small RNA (S-RNA), which codes for the nucleoprotein (NP) and the glycoprotein precursor (GPC), was followed in L and MDCK cells after infection with multiplicities (m.o.i.) ranging from 0.01 to 100. In L cells, the yields of both plaque-forming units and interfering particles varied inversely with the m.o.i. Northern blot analysis revealed that early after infection with high multiplicity NP-mRNA was present, but later few or no signals of any specificity were registered. After low m.o.i. the results were negative at 8 hr, but large quantities of mRNAs for NP and GPC as well as viral genomic S-RNA and genomic-sized complementary S-RNA had been synthesized at 48 hr. In MDCK cells, throughout the range of m.o.i. both entities attained lower levels and most were generated at m.o.i. one. The degree of hybridization correlated roughly with the quantity of infectious virus to which the cells had been exposed. In the cells of both lines the NP-mRNA corresponded to the synthesis of its translation product, but once produced, most of it appeared to be retained in the phosphorylated form. We assume that the homologous interference seen in L cells after infection with high m.o.i. results from a host-dependent inhibition of viral transcription and replication mediated by NP.

Lymphocytic choriomeningitis virus: IX. Properties of the nucleocapsid

Nucleocapsids (NC) of lymphocytic choriomeningitis (LCM) virus were obtained by treatment of purified infectious virus with detergent and salt at high concentrations, followed by gradient centrifugation. NC thus prepared contained 31 S RNA, 23 S RNA, and a protein with an apparent mol wt 63,000 and an isoelectric point pH 5.7 (p63), assumed to be the nucleoprotein. We had previously observed that the intact LCM virus contained two kinds of protein with mol wt 63,000 separable by their isoelectric points which were pH 5.7 and 7.7, respectively. We now found that the latter component was removed from the NC together with the glycoproteins, and labeling studies revealed that it was closely associated with the viral envelope. We have named the protein with a mol wt of 63,000 and an isoelectric point pH 7.7 p63E (E for envelope). By limited proteolysis both viral components of mol wt 63,000 could not be distinguished, indicating that they are basically identical. It appears that p63E is the phosphorylated form of p63.

Lymphocytic choriomeningitis virus. X. Demonstration of nucleoprotein on the surface of infected cells.

Of a total of 17 monoclonal antibodies (MAb) directed against structural proteins of the lymphocytic choriomeningitis (LCM) virus, 3 were specific for the viral nucleoprotein (p63) and attached to the plasma membrane of infected cells, as disclosed by the indirect immunofluorescence procedure and complement-mediated cytolysis. We had previously demonstrated that a portion of the nucleoprotein (p63E) was part of the envelope of the intact virion (M. Bruns, W. Zeller, H. Rohdewohld, and F. Lehmann-Grube (1986) Virology 151, 77-85), and we now show that after external iodination of virions followed by limited proteolysis the label was attached to the smallest peptide thus obtained. If purified nucleocapsids were labeled with 125I, digested as before, and incubated with an anti-p63 MAb that has the ability to bind the surface of the infected cell, a similarly small peptide was precipitated; an antibody specific for p63 but not recognizing it on the cell surface precipitated the largest peptide and failed to bring down the small one. We conclude that the epitopes complementary to a few of our anti-p63 MAb are represented on both the virion and the surface of virus-infected cells.

Lymphocytic choriomeningitis virus. V. Proposed structural arrangement of proteins in the virion.

Previous work by M.J. Buchmeier and his colleagues and by our group has led to the conclusion that the lymphocytic choriomeningitis (LCM) virus contains seven distinct structural proteins, which we have named p200, gp85, p77, p63, gp60, gp44, and gp35. Their arrangement in the virion has now been analysed by establishing nearest-neighbour relationships with a homobifunctional crosslinker, by performing polyacrylamide gel electrophoresis in parallel under reducing and non-reducing conditions, and by determining the proteins that are covalently bound to viral lipids. A hypothetical model of the virion of LCM virus is proposed. Its envelope is assumed to consist of a membranous layer composed of gp60 and lipids and two types of spikes with either gp85 or gp44 as tips and gp35 as bases. The last-mentioned glycoprotein also appears to be complexed with p63, the main protein component of the nucleocapsid, and this in turn was found to be spatially associated with p200. Probably p77 is also an internal component, but a more exact position cannot yet be assigned to this protein.

Nucleoprotein-specific nonneutralizing antibodies speed up LCMV elimination independently of complement and FcγR.

CD8+ T cells have an essential role in controlling lymphocytic choriomeningitis virus (LCMV) infection in mice. Here, we examined the contribution of humoral immunity, including nonneutralizing antibodies (Abs), in this infection induced by low virus inoculation doses. Mice with impaired humoral immunity readily terminated infection with the slowly replicating LCMV strain Armstrong but showed delayed virus elimination after inoculation with the faster replicating LCMV strain WE and failed to clear the rapidly replicating LCMV strain Docile, which is in contrast to the results obtained with wild‐type mice. Thus, the requirement for adaptive humoral immunity to control the infection was dependent on the replication speed of the LCMV strains used. Ab transfers further showed that LCMV‐specific IgG Abs isolated from LCMV immune serum accelerated virus elimination. These Abs were mainly directed against the viral nucleoprotein (NP) and completely lacked virus neutralizing activity. Moreover, mAbs specific for the LCMV NP were also able to decrease viral titers after transfer into infected hosts. Intriguingly, neither C3 nor Fcγ receptors were required for the antiviral activity of the transferred Abs. In conclusion, our study suggests that rapidly generated nonneutralizing Abs specific for the viral NP speed up virus elimination and thereby may counteract T‐cell exhaustion.

Isolation of a glycoprotein and two structural proteins of Maedi-Visna virus.

Abstract A viral glycoprotein of Maedi-Visna virus was purified by affinity chromatography, rate zonal centrifugation, and isoelectric focusing. Furthermore, two structural proteins of the virus were purified by isoelectric focusing and Sephadex gel filtration. In sodium dodecyl sulfate—polyacrylamide gel electrophoresis they were found to be homogeneous proteins with relative molecular weights of 15,000 (p15), 24,000 (p24), and 70,000 for the viral glycoprotein (gp70). In 71 out of 90 sera from sheep infected with Maedi-Visna, antibodies against gp70 were found, and only in five sera could antibodies be detected against p24 by agar gel immunodiffusion tests.

Entry of Duck Hepatitis B Virus into Primary Duck Liver and Kidney Cells after Discovery of a Fusogenic Region within the Large Surface Protein

ABSTRACT Hepatitis B viruses exhibit a narrow host range specificity that is believed to be mediated by a domain of the large surface protein, designated L. For duck hepatitis B virus, it has been shown that the pre-S domain of L binds to carboxypeptidase D, a cellular receptor present in many species on a wide variety of cell types. Nonetheless, only hepatocytes become infected. It has remained vague which viral features determine host range specificity and organotropicity. By using chymotrypsin to treat duck hepatitis B virus, we addressed the question of whether a putative fusogenic region within the amino-terminal end of the small surface protein may participate in viral entry and possibly constitute one of the determinants of the host range of the virus. Addition of the enzyme to virions resulted in increased infectivity. Remarkably, even remnants of enzyme-treated subviral particles proved to be inhibitory to infection. A noninfectious deletion mutant devoid of the binding region for carboxypeptidase D could be rendered infectious for primary duck hepatocytes by treatment with chymotrypsin. Although because of the protease treatment mutant and wild-type viruses may have become infectious in an unspecific and receptor-independent manner, their host range specificity was not affected, as shown by the inability of the virus to replicate in different hepatoma cell lines, as well as primary chicken hepatocytes. Instead, the organotropicity of the virus could be reduced, which was demonstrated by infection of primary duck kidney cells.

Lymphocytic choriomeningitis virus. VII. Structural alterations of the virion by treatment with proteolytic enzymes without loss of infectivity.

Treatment of lymphocytic choriomeningitis virus with proteolytic enzymes, hyaluronidase, and phospholipase C increased infectious titres. Biochemical analysis of bromelain- and trypsin-treated virus revealed that infectivity was high in spite of the decrease to low or undetectable levels of all viral glycoproteins as well as partial degradation of the nucleoprotein.

Characterization of a virus variant produced by L cells persistently infected with lymphocytic choriomeningitis virus

Continuous cultivation of murine L cells infected with lymphocytic choriomeningitis virus strain Armstrong leads to production of L(Arm) cells, which produce a predominantly cell-associated attenuated variant, the L(Arm) virus. The relatively few infectious particles that are released have lost the ability to form plaques on L cells and to cause illness in mice even if inoculated intracerebrally. Based on equal protein M(r)s, antigenicity and protein kinase activity, essentially identical results were obtained for the purified Armstrong and L(Arm) viruses. There was also no difference in production and release of particles with the potential to cause homologous interference. Such particles consisted of two types, one of which was highly susceptible to u.v.-irradiation, the other was highly resistant. In the case of the L(Arm) virus interfering particles, it appears that the u.v.-irradiation-susceptible forms represented infectious virus. Purified L(Arm) virus particles contained considerable quantities of subgenomic forms of (small) S- and (large) L-RNA and their complementary counterparts, which all appeared to be replicated autonomously in an unenriched manner.