Induction of an HPV 6bL1-specific mucosal IgA response by DNA immunization.

Human papillomavirus (HPV) plays a crucial role in the development of human anogenital dysplasia. To prevent infection, it is important to induce an HPV-specific mucosal immune response. We investigated whether DNA vaccination would induce an intravaginal mucosal antibody response against HPV 6bL1. New Zealand White rabbits were immunized with an HPV 6bL1 DNA vaccine by one of the three routes: muscular, vaginal, or rectal. We found that vaginal immunization of rabbits with HPV 6bL1 DNA induced 6bL1 virus-like particle-specific lgA antibodies in vaginal secretions. They were detectable until at least 14 weeks after the first immunization. The antibodies also showed neutralizing activity in a hemagglutination inhibition assay. No mucosal immune response was detected in vaginal secretions of rabbits immunized intramuscularly or intrarectally. Our data suggest that vaginal immunization with HPV 6bL1 DNA induces long-lasting IgA responses with neutralizing activity in vaginal secretions of rabbits.

Identification of mouse hepatitis virus papain-like proteinase 2 activity.

Mouse hepatitis virus (MHV) is a 31-kb positive-strand RNA virus that is replicated in the cytoplasm of infected cells by a viral RNA-dependent RNA polymerase, termed the replicase. The replicase is encoded in the 5'-most 22 kb of the genomic RNA, which is translated to produce a polyprotein of >800 kDa. The replicase polyprotein is extensively processed by viral and perhaps cellular proteinases to give rise to a functional replicase complex. To date, two of the MHV replicase-encoded proteinases, papain-like proteinase 1 (PLP1) and the poliovirus 3C-like proteinase (3CLpro), have been shown to process the replicase polyprotein. In this report, we describe the cloning, expression, and activity of the third MHV proteinase domain, PLP2. We show that PLP2 cleaves a substrate encoding the first predicted membrane-spanning domain (MP1) of the replicase polyprotein. Cleavage of MP1 and release of a 150-kDa intermediate, p150, are likely to be important for embedding the replicase complex in cellular membranes. Using an antiserum (anti-D11) directed against the C terminus of the MP1 domain, we verified that p150 encompasses the MP1 domain and identified a 44-kDa protein (p44) as a processed product of p150. Pulse-chase experiments showed that p150 is rapidly generated in MHV-infected cells and that p44 is processed from the p150 precursor. Protease inhibitor studies revealed that unlike 3CLpro activity, PLP2 activity is not sensitive to cysteine protease inhibitor E64d. Furthermore, coexpression studies using the PLP2 domain and a substrate encoding the MP1 cleavage site showed that PLP2 acts efficiently in trans. Site-directed mutagenesis studies confirmed the identification of cysteine 1715 as a catalytic residue of PLP2. This study is the first to report enzymatic activity of the PLP2 domain and to demonstrate that three distinct viral proteinase activities process the MHV replicase polyprotein.

Colocalization and membrane association of murine hepatitis virus gene 1 products and De novo-synthesized viral RNA in infected cells.

Murine hepatitis virus (MHV) gene 1, the 22-kb polymerase (pol) gene, is first translated into a polyprotein and subsequently processed into multiple proteins by viral autoproteases. Genetic complementation analyses suggest that the majority of the gene 1 products are required for viral RNA synthesis. However, there is no physical evidence supporting the association of any of these products with viral RNA synthesis. We have now performed immunofluorescent-staining studies with four polyclonal antisera to localize various MHV-A59 gene 1 products in virus-infected cells. Immunoprecipitation experiments showed that these antisera detected proteins representing the two papain-like proteases and the 3C-like protease encoded by open reading frame (ORF) 1a, the putative polymerase (p100) and a p35 encoded by ORF 1b, and their precursors. De novo-synthesized viral RNA was labeled with bromouridine triphosphate in lysolecithin-permeabilized MHV-infected cells. Confocal microscopy revealed that all of the viral proteins detected by these antisera colocalized with newly synthesized viral RNA in the cytoplasm, particularly in the perinuclear region of infected cells. Several cysteine and serine protease inhibitors, i.e., E64d, leupeptin, and zinc chloride, inhibited viral RNA synthesis without affecting the localization of viral proteins, suggesting that the processing of the MHV gene 1 polyprotein is tightly associated with viral RNA synthesis. Dual labeling with antibodies specific for cytoplasmic membrane structures showed that MHV gene 1 products and RNA colocalized with the Golgi apparatus in HeLa cells. However, in murine 17CL-1 cells, the viral proteins and viral RNA did not colocalize with the Golgi apparatus but, instead, partially colocalized with the endoplasmic reticulum. Our results provide clear physical evidence that several MHV gene 1 products, including the proteases and the polymerase, are associated with the viral RNA replication-transcription machinery, which may localize to different membrane structures in different cell lines.

Processing of the coronavirus MHV-JHM polymerase polyprotein: identification of precursors and proteolytic products spanning 400 kilodaltons of ORF1a.

The replicase of mouse hepatitis virus strain JHM (MHV-JHM) is encoded by two overlapping open reading frames, ORF1a and ORF1b, which are translated to produce a 750-kDa precursor polyprotein. The polyprotein is proposed to be processed by viral proteinases to generate the functional replicase complex. To date, only the MHV-JHM amino-terminal proteins p28 and p72, which is processed to p65, have been identified. To further elucidate the biogenesis of the MHV-JHM replicase, we cloned and expressed five regions of ORF1a in bacteria and prepared rabbit antisera to each region. Using the immune sera to immunoprecipitate radiolabeled proteins from MHV-JHM infected cells, we determined that the MHV-JHM ORF1a is initially processed to generate p28, p72, p250, and p150. Pulse-chase analysis revealed that these intermediates are further processed to generate p65, p210, p40, p27, the MHV 3C-like proteinase, and p15. A putative replicase complex consisting of p250, p210, p40, p150, and a large protein (> 300 kDa) coprecipitate from infected cells disrupted with NP-40, indicating that these proteins are closely associated even after initial proteolytic processing. Immunofluorescence studies revealed punctate labeling of ORF1a proteins in the perinuclear region of infected cells, consistent with a membrane-association of the replicase complex. Furthermore, in vitro transcription/translation studies of the MHV-JHM 3Cpro and flanking hydrophobic domains confirm that 3C protease activity is significantly enhanced in the presence of canine microsomal membranes. Overall, our results demonstrate that the MHV-JHM ORF1a polyprotein: (1) is processed into more than 10 protein intermediates and products, (2) requires membranes for efficient biogenesis, and (3) is detected in discrete membranous regions in the cytoplasm of infected cells.

Sequence close to the N-terminus of L2 protein is displayed on the surface of bovine papillomavirus type 1 virions.

The bovine papillomavirus type 1 (BPV1) L2 protein purified from Escherichia coli was used as an antigen to produce monoclonal antibodies (MAbs). A total of 26 individual clones which recognized the BPV1 L2 protein were obtained. Using infectious BPV1 virus particles, 3 of the MAbs were found to interact with BPV1 virus particles. Binding of the MAbs to BPV1 was confirmed by immunoelectron microscopy. A set of 92 13-mer peptides overlapping by 8 amino acids spanning the entire BPV1 L2 protein was synthesized on a membrane and used to map the epitopes recognized by these antibodies. Seventeen linear epitopes were identified. Our results revealed that a sequence toward the N-terminus of the L2 protein (aa 61-123) is displayed on the virus surface, while the remaining L2 sequences are hidden inside the virus capsid. Although the polyclonal antisera raised against BPV1 L2 neutralized the BPV1 virus, we failed to detect any neutralizing activity for the 3 L2-specific monoclonal antibodies which bound to the BPV1 particles. This suggests that extra binding sites may be needed for neutralization. This study prompted us to propose a model about how L1 and L2 proteins may interact during infectious papillomavirus assembly.

Lack of augmenting effect of interferon-gamma on dengue virus multiplication in human peripheral blood monocytes.

The effect of interferon-gamma (IFN-gamma) on dengue virus multiplication in human peripheral blood monocytes was investigated. Enriched monocytes were treated with IFN-gamma and then infected with dengue virus type 2 either directly or in the presence of optimal infection-enhancing levels of antibodies. Pretreatment of monocytes from dengue-immune donors with 100 IU/ml of IFN-gamma caused 12- to 97-fold and 13- to 137-fold reduction of virus yields at 24 hr after infection in the absence and presence of an anti-flavivirus monoclonal antibody, respectively. IFN-gamma also diminished virus yields when infection of monocytes from a donor who lacked anti-dengue antibody was enhanced 40-fold. The percentage of infected monocytes in IFN-gamma-pretreated cultures was similarly reduced. Dominance of the antiviral effect of IFN-gamma in monocytes is in contrast to an augmenting effect previously observed in the promonocytic cell line U937.