Human metapneumovirus (hMPV) is a member of the Pneumovirinea subfamily within the Paramyxoviridea family. Since its discovery in 2001, hMPV has been isolated in several continents, which suggests its prevalence worldwide. hMPV resembles human respiratory syncytial virus with regard to disease symptoms and its ability to infect and cause disease in young infants as well as individuals of all ages. The aim of the current study was to construct an efficient high-level yeast expression system for the generation of hMPV nucleocapsid (N) protein and to develop monoclonal antibodies (MAbs) suitable for hMPV detection. The genome of hMPV was isolated from oral fluid of an infected patient by using specific primers and reverse transcriptase polymerase chain reaction (RT-PCR). DNA sequence corresponding to the N protein gene was inserted into yeast expression vector under inducible GAL7 promoter. SDS-PAGE analysis of crude lysates of yeast Saccharomyces cerevisiae harbouring recombinant plasmid revealed the presence of a protein band of approximately 43 kDa corresponding to the molecular weight of hMPV N protein. Electron microscopy analysis of purified N protein revealed nucleocapsid-like structures with typical herring-bone morphology: rods of 20 nm diameter with repeated serration along the edges and central core of 5 nm. Recombinant hMPV N protein was reactive with human serum specimens collected from patients with confirmed hMPV infection. After immunization of mice with recombinant hMPV N protein, a panel of MAbs was generated. The specificity of newly generated MAbs was proven by immunofluorescence analysis of hMPV-infected cells. Epitope mapping using truncated variants of hMPV N revealed localization of linear MAb epitopes at the N-terminus of hMPV N protein, between amino acid residues 1 and 90. The MAbs directed against conformational epitopes did not recognize hMPV N protein variants containing either N- or C-terminal truncations. The reactivity of recombinant hMPV N protein with hMPV-positive serum specimens and the ability of MAbs to recognize virus-infected cells confirms the antigenic similarity between yeast-expressed hMPV N protein and native viral nucleocapsids. In conclusion, recombinant hMPV N protein and hMPV-specific MAbs provide new diagnostic reagents for hMPV infection.

The coronavirus nucleocapsid (N) is a multifunctional phosphoprotein that encapsidates the genomic RNA into a helical nucleocapsid within the mature virion. The protein also plays roles in viral RNA transcription and/or replication and possibly viral mRNA translation. Phosphorylation is one of the most common post-translation modifications that plays important regulatory roles in modulating protein functions. It has been speculated for sometime that phosphorylation could play an important role in regulation of coronavirus N protein functions. As a first step toward positioning to address this we have identified the amino acids that are phosphorylated on the mouse hepatitis coronavirus (MHV) A59 N protein. High performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) was used to identify phosphorylated sites on the N protein from both infected cells and purified extracellular virions. A total of six phosphorylated sites (S162, S170, T177, S389, S424 and T428) were identified on the protein from infected cells. The same six sites were also phosphorylated on the extracellular mature virion N protein. This is the first identification of phosphorylated sites for a group II coronavirus N protein.

The coronavirus disease 2019 (COVID-19) pandemic is fading, however its etiologic agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues posing - despite the availability of licensed vaccines - a global health threat, due to the potential emergence of vaccine-resistant SARS-CoV-2 variants. This makes the development of new drugs against COVID-19 a persistent urgency and sets as research priority the validation of novel therapeutic targets within the SARS-CoV-2 proteome. Among these, a promising one is the SARS-CoV-2 nucleocapsid (N) phosphoprotein, a major structural component of the virion with indispensable role in packaging the viral genome into a ribonucleoprotein (RNP) complex, which also contributes to SARS-CoV-2 innate immune evasion by inhibiting the host cell type-I interferon (IFN-I) response. By combining miniaturized differential scanning fluorimetry with microscale thermophoresis, we found that the 100-year-old drug Suramin interacts with SARS-CoV-2 N-terminal domain (NTD) and C-terminal domain (CTD), thereby inhibiting their single-stranded RNA (ssRNA) binding function with low-micromolar Kd and IC50 values. Molecular docking suggests that Suramin interacts with basic NTD cleft and CTD dimer interface groove, highlighting three potentially druggable ssRNA binding sites. Electron microscopy shows that Suramin inhibits the formation in vitro of RNP complex-like condensates by SARS-CoV-2 N with a synthetic ssRNA. In a dose-dependent manner, Suramin also reduced SARS-CoV-2-induced cytopathic effect on Vero E6 and Calu-3 cells, partially reverting the SARS-CoV-2 N-inhibited IFN-I production in 293T cells. Our findings indicate that Suramin inhibits SARS-CoV-2 replication by hampering viral genome packaging, thereby representing a starting model for design of new COVID-19 antivirals.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly pathogenic Baculoviridae that targets insects, whose core gene, ac81, has an unknown function. To determine the role of ac81 in the life cycle of AcMNPV, an ac81-knockout (Ac-81KO-GP) was constructed through homologous recombination in Escherichia coli. We determined that no budded virions were produced in Ac-81KO-GP-transfected Sf9 cells, while there was no effect on viral DNA replication. Electron microscopy (EM) analysis revealed that occlusion-derived virions (ODVs) envelopment and the subsequent embedding of virions into occlusion bodies (OBs) were aborted due to ac81 deletion. Interestingly, confocal microscopy and immunofluorescence analysis revealed that Ac81 was predominantly localized to the ring zone of nuclei during the late phase of infection. In addition, Ac81 was localized to the mature and premature ODVs in virus-infected cells within the ring zone as revealed by immuno-electron microscopy (IEM) analysis. Furthermore, we determined that Ac81 contained a functional hydrophobic transmembrane (TM) domain, whose deletion resulted in a phenotype similar to that of Ac-81KO-GP. These results suggest that Ac81 might be a TM protein that played an important role in nucleocapsid envelopment.

Porcine deltacoronavirus (PDCoV) is a newly discovered enterotropic swine coronavirus that causes enteritis and diarrhea in piglets. Like other coronaviruses, PDCoV commonly contains 4 major structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. Among these, the N protein is known to be the most abundant and multifunctional viral component. Therefore, as the first step toward understanding the biology of PDCoV, the present study investigated functional characteristics and expression dynamics of host proteins in a stable porcine cell line constitutively expressing the PDCoV N protein. Similar to N proteins of other coronaviruses, the PDCoV N protein was found to interact with itself to form non-covalently linked oligomers and was mainly localized to the nucleolus. We then assessed alterations in production levels of proteins in the N-expressing PK (PK-PDCoV-N) cells at different time points by means of proteomic analysis. According to the results of high-resolution two-dimensional gel electrophoresis, a total of 43 protein spots were initially found to be differentially expressed in PK-PDCoV-N cells in comparison with control PK cells. Of these spots, 10 protein spots showed a statistically significant alteration, including 8 up-regulated and 2 down-regulated protein spots and were picked for subsequent protein identification by peptide mass fingerprinting following matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The affected cellular proteins that we identified in this study were classified into the functional groups involved in various cellular processes such as cell division, metabolism, the stress response, protein biosynthesis and transport, cytoskeleton networks and cell communication. Notably, two members of the heat shock protein 70 family were found to be up-regulated in PK-PDCoV-N cells. These proteomic data will provide insights into the specific cellular response to the N protein during PDCoV infection.

Yellow head virus (YHV) is an invertebrate nidovirus that is highly pathogenic for marine shrimp. Nucleotide sequence analysis indicated that the YHV ORF2 gene encodes a basic protein (pI = 9.9) of 146 amino acids with a predicted molecular weight of 16,325.5 Da. The deduced amino acid sequence indicated a predominance of basic (15.1%), acidic (9.6%) and hydrophilic polar (34.3%) residues and a high proportion proline and glycine residues (16.4%). The ORF2 gene was cloned and expressed in Escherichia coli as a M(r) = 21 kDa His(6)-protein that reacted with YHV nucleoprotein (p20) monoclonal antibody. Segments representing the four linear quadrants of the nucleoprotein were also expressed in E. coli as GST-fusion proteins. Immunoblot analysis using YHV polyclonal rabbit antiserum indicated the presence of linear epitopes in all except the V(37)-Q(74) quadrant. Immunoblot analysis of the GST-fusion proteins and C-terminally truncated segments of the nucleoprotein allowed mapping of YHV monoclonal antibodies Y19, Y20 and YII4 to linear epitopes in the acidic domain between amino acids I(116) and E(137). The full-length nucleoprotein was expressed at high level in E. coli and was easily purified in quantity from the soluble cell fraction by Ni(+)-NTA affinity chromatography.

A novel coronavirus (CoV) has recently been identified as the aetiological agent of severe acute respiratory syndrome (SARS). Nucleocapsid (N) proteins of the Coronaviridae family have no discernable homology, but they share a common nucleolar-cytoplasmic distribution pattern. There are three putative nuclear localization signal (NLS) motifs present in the N. To determine the role of these putative NLSs in the intracellular localization of the SARS-CoV N, we performed a confocal microscopy analysis using rabbit anti-N antisera. In this report, we show that the wild type N was distributed mainly in the cytoplasm. The N-terminal of the N, which contains the NLS1 (aa38-44), was localized to the nucleus. The C-terminus of the N, which contains both NLS2 (aa257-265) and NLS3 (aa369-390) was localized to the cytoplasm and the nucleolus. Results derived from analysis of various deletion mutations show that the region containing amino acids 226-289 is able to mediate nucleolar localization. The deletion of two hydrophobic regions that flanked the NLS3 recovered its activity and localized to the nucleus. Furthermore, deletion of leucine rich region (220-LALLLLDRLNRL) resulted in the accumulation of N to the cytoplasm and nucleolus, and when fusing this peptide to EGFP localization was cytoplasmic, suggesting that the N may act as a shuttle protein. Differences in nuclear/nucleolar localization properties of N from other members of coronavirus family suggest a unique function for N, which may play an important role in the pathogenesis of SARS.

Porcine deltacoronavirus (PDCoV) is an emerging coronavirus that causes vomiting, diarrhea, dehydration, and even death of piglets, resulting in significant losses to the pig industry worldwide. However, the epitopes of PDCoV remain largely unknown. In this study, a monoclonal antibody (mAb) against the PDCoV nucleocapsid (N) protein, termed 9G1, was prepared using the lymphocyte hybridoma technique, and was identified as a type IgG1 with a κ light chain and reacted with the native N protein of PDCoV. Furthermore, the epitope recognized by the 9G1 mAb was subjected to western blot and an ELISA using truncated recombinant proteins and synthetic polypeptides of the PDCoV N protein. The results indicate that 9G1 mAb recognized the epitope, G59TPIPPSYAFYY70 (EP-9G1), a novel linear B cell epitope of the PDCoV N protein. A comparison analysis revealed that the EP-9G1 epitope was highly conserved among PDCoV strains, in which four residues (G59-F68YY70) were observed among different coronavirus genera. These data demonstrate that the EP-9G1 epitope identified in this study provides some basic information for further characterization of the antigenic structure of the PDCoV N protein and has potential use for developing diagnostic reagents for PDCoV.

The HIV-1 nucleocapsid protein (NC) is considered as an emerging drug target for the therapy of AIDS. Several studies have highlighted the crucial role of NC within the viral replication cycle. However, although NC inhibition has provided in vitro and in vivo antiretroviral activity, drug-candidates which interfere with NC functions are still missing in the therapeutic arsenal against HIV. Based on previous studies, where the dynamic behavior of NC and its ligand binding properties have been investigated by means of computational methods, here we used a virtual screening protocol for discovering novel antiretroviral compounds which interact with NC. The antiretroviral activity of virtual hits was tested in vitro, whereas biophysical studies elucidated the direct interaction of most active compounds with NC(11-55), a peptide corresponding to the zinc finger domain of NC. Two novel antiretroviral small molecules capable of interacting with NC are presented here.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRSV, has ranked among the most economically important veterinary infectious diseases globally. Recently, tripartite motif (TRIMs) family members have arisen as novel restriction factors in antiviral immunity. Noteworthy, TRIM26 was reported as a binding partner of IRF3, TBK1, TAB1, and NEMO, yet its role in virus infection remains controversial. Herein, we showed that TRIM26 bound N protein by the C-terminal PRY/SPRY domain. Moreover, ectopic expression of TRIM26 impaired PRRSV replication and induced degradation of N protein. The anti-PRRSV activity was independent of the nuclear localization signal (NLS). Instead, deletion of the RING domain, or the PRY/SPRY portion, abrogated the antiviral function. Finally, siRNA depletion of TRIM26 resulted in enhanced production of viral RNA and virus yield in porcine alveolar macrophages (PAMs) after PRRSV infection. Overexpression of an RNAi-resistant TRIM26 rescue-plasmid led to the acquisition of PRRSV restriction in TRIM26-knockdown cells. Together, these data add TRIM26 as a potential target for drug design against PRRSV.

Avian pneumovirus (APV) has recently been described as the cause of a new respiratory syndrome in turkey flocks in the United States. We here describe the complete sequence of the nucleocapsid (N) and phosphoprotein (P) genes of this emerging APV (APV/US). Our results show 59 and 61% nucleotide sequence identity of the APV/US N gene with N genes of previously described European APV subgroups A and B, respectively. The P gene of APV/US showed only 53% nucleotide sequence identity with the ortholog from APV subgroup A. Phylogenetic analyses of both N and P genes clearly demonstrate that the APV/US lineage is evolutionarily related but distinct from European APVs. Moreover, sequence analysis of the N and P genes from two laboratory adapted isolates of APV/US (APV/MN-1a and APV/MN-1b) and from ten clinical samples from APV-infected turkeys suggests only modest level of amino acid divergence in the N (0-0.3%) and P (0-1.4%) proteins. Taken together, the results of this study indicate support that APV/US represents a new subgroup (subgroup C) of APV and show that there is limited heterogeneity in the N and P genes of APV/US isolates.

In order to investigate the genomic organization of the single-nucleocapsid nucleopolyhedrovirus (SNPV) of Buzura suppressaria (BusuNPV), the HindIII-I fragment located at map units (mu) 26.6-29.4 of the viral genome was sequenced. The fragment contained two partial and three complete open reading frames (ORFs) representing the 3' end of a polyhedron envelope protein gene (pep), a homologue of the AcMNPV ORF117, a conotoxin-like protein gene (ctl), an inhibitor of apoptosis gene (iap) and a superoxide dismutase gene (sod), respectively. These five genes were identified for the first time in a SNPV. Sequence analysis further revealed that these ORFs have the same conserved motifs and gene structure as those observed in their homologues from other baculoviruses. Between ctl and iap, an intergenic region of about 700 basepairs with structure similar to non-hr origins of DNA replication was observed. The genomic arrangement of the ORFs in the BusuNPV HindIII-I fragment is very different from the arrangement of their homologues in the genome of Autographa californica multiple nucleocapsid (M) NPV and other baculoviruses to date. Our data suggest that on the basis of gene arrangement, BusuNPV belongs to a distinct taxon within the Baculoviridae family, corroborating our previous conclusions derived from phylogeny analysis of several BusuNPV genes.

The emergency SARS-CoV-2, a member of severe acute respiratory syndrome-related coronaviruses (SARSr-CoV), is still greatly harming the health of mankind. SARS-CoV-2-specific monoclonal antibodies (MAbs), which can identify SARS-CoV-2 from common human coronaviruses, are considered to extensively apply to developing rapid and reliable antigen assays. In this study we generated a rabbit MAb (RAb) detecting SARS-CoV-2 nucleocapsid protein (NP), which has cross-reaction with SARS-CoV-1 NP, but not with NPs of MERS and common human CoVs (OC43, NL63, 229E, and HKU1). With truncated NP fragments and synthesized peptides, the linear epitope detected by RAb was mapped in peptide N4-8, 393-407 amino acid residue (TLLPAADLDDFSKQL) of SARS-CoV-2 NP. This epitope N4-8 was highly conserved in SARSr-CoVs, including SARS-CoV-2, SARS-CoV-1, and bat CoV RaTG13 strain. However, the corresponding peptide of bat SARSr-CoV BtKY72 strain could not be recognized by RAb, which indicates amino acid D399 may be critical for N4-8 epitope detected by RAb. The present study will be conducive to developing reliable diagnosis for SARS-CoV-2 and gaining insights into the function of the SARS-CoV-2 N protein.

Breda virus (BRV), a member of the genus torovirus, is an established etiological agent of diarrhea of cattle, which is found as two separate serotypes, BRV-1 and BRV-2. In this study, a 7.5 kb fragment of the BRV-1 genome that bracketed the genes for the structural proteins of BRV was amplified by long RT-PCR and the amplicon purified and sequenced directly. Sequence analysis revealed the presence of four open reading frames (ORF) corresponding to the peplomer (S), envelope (M), and nucleocapsid (N) genes, and an ORF for a novel 1.2 kb gene located between the M and N genes. This new gene was identical in nucleotide sequence to the hemagglutinin-esterase (HE) gene of BRV-2. With the exception of this new ORF, BRV-1 manifests 80% nucleotide sequence identity with the torovirus prototype, Berne virus (BEV) in the 7.5 kb region from the 3' end of the genome that contains the genes for the structural proteins. A 504 base segment containing the ORF for the BRV-1 N gene was amplified by RT-PCR, and cloned into an Escherichia coli expression system. The resulting protein was purified by SDS-PAGE and used to immunize guinea pigs. Hyperimmune serum was reactive with bovine torovirus (BTV) and human torovirus (HTV) antigens. By immunoelectron microscopy, it was shown to aggregate broken but not intact torovirus particles from BTV-positive fecal specimens. By immunoblot, the hyperimmune serum reacted specifically with the 20 kD N proteins of both BTV and HTV, as well as with the expressed N protein. BRV-1 and BRV-2 immune sera from gnotobiotic calves, but not human convalescent sera from HTV-infected patients, reacted with the expressed N protein by immunoblot. These findings were applied to the design of a dot blot assay that could specifically detect BTV and HTV from fecal specimens.

A member of the family of coronaviruses has previously been identified as the cause of the severe acute respiratory syndrome (SARS). In this study, several monoclonal antibodies against the nucleocapsid protein have been generated to examine distribution of the nucleocapsid in virus-infected cells and to study antigenic regions of the protein. Confocal microscopic analysis identified nucleocapsids packaged in vesicles in the perinuclear area indicating viral synthesis at the endoplasmic reticulum and Golgi apparatus. The monoclonal antibodies bound to the central and carboxyterminal half of the nucleocapsid protein indicating prominent exposure and immunogenicity of this part of the protein. Antibodies recognised both linear and conformational epitopes. Predictions of antigenicity using mathematical modelling based on hydrophobicity analysis of SARS nucleoprotein could not be confirmed fully. Antibody binding to discontinuous peptides provides evidence that amino acids 274-283 and 373-382 assemble to a structural unit particularly rich in basic amino acids. In addition, amino acids 286-295, 316-325 and 361-367 that represent the epitope recognised by monoclonal antibody 6D11C1 converge indicating a well-structured C-terminal region of the SARS virus nucleocapsid protein and functional relationship of the peptide regions involved. Alternatively, dimerisation of the nucleocapsid protein may result in juxtaposition of the amino acid sequences 316-325 and 361-367 on one nucleoprotein molecule to amino acid 286-295 on the second peptide. The monoclonal antibodies will be available to assess antigenicity and immunological variabilities between different SARS CoV strains.

Severe acute respiratory syndrome-coronavirus nucleocapsid (SARS-CoV N) protein has been found to be important to the processes related to viral pathogenesis, such as virus replication, interference of the cell process and modulation of host immune response; detection of the antigen has been used for the early diagnosis of infection. We have used recombinant N protein expressed in insect cells to generate 17 mAbs directed against this protein. We selected five mAbs that could be used in various diagnostic assays, and all of these mAbs recognized linear epitopes. Three IgG(2b) mAbs were recognized within the N-terminus of N protein, whereas the epitope of two IgG(1) mAbs localized within the C-terminus. These mAbs were found to have significant reactivity with both non-phosphorylated and phosphorylated N proteins, which resulted in high reactivity with native N protein in virus-infected cells; however, they did not show cross-reactivity with human coronavirus. Therefore, these results suggested that these mAbs would be useful in the development of various diagnostic kits and in future studies of SARS-CoV pathology.

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein plays an essential role in several stages of HIV-1 replication. One important function of HIV-1 NC is to act as a nucleic acid chaperone, in which the protein facilitates nucleic acid rearrangements important for reverse transcription and recombination. NC contains only 55 amino acids, with 15 basic residues and two zinc fingers, each having a single aromatic residue (Phe16 and Trp37). Despite its simple structure, HIV-1 NC appears to have optimal chaperone activity, including the ability to strongly aggregate nucleic acids, destabilize nucleic acid secondary structure, and facilitate rapid nucleic acid annealing. Here we combine single molecule DNA stretching experiments with ensemble solution studies of protein-nucleic acid binding affinity, oligonucleotide annealing, and nucleic acid aggregation to measure the characteristics of wild-type (WT) and aromatic residue mutants of HIV-1 NC that are important for nucleic acid chaperone activity. These in vitro results are compared to in vivo HIV-1 replication for viruses containing the same mutations. This work allows us to directly relate HIV-1 NC structure with its function as a nucleic acid chaperone in vitro and in vivo. We show that replacement of either aromatic residue with another aromatic residue results in a protein that strongly resembles WT NC. In contrast, single amino acid substitutions of either Phe16Ala or Trp37Ala significantly slow down NC's DNA interaction kinetics, while retaining some helix-destabilization capability. A double Phe16Ala/Trp37Ala substitution further reduces the latter activity. Surprisingly, the ensemble nucleic acid binding, annealing, and aggregation properties are not significantly altered for any mutant except the double aromatic substitution with Ala. Thus, elimination of a single aromatic residue from either zinc finger strongly reduces NC's chaperone activity as determined by single molecule DNA stretching experiments without significantly altering its ensemble-averaged biochemical properties. Importantly, the substitution of aromatic residues with Ala progressively decreases NC's nucleic acid chaperone activity while also progressively inhibiting viral replication. Taken together, these data support the critical role of HIV-1 NC's aromatic residues, and establish a direct and statistically significant correlation between nucleic acid chaperone activity and viral replication.

A swine acute diarrhea syndrome coronavirus (SADS-CoV) that causes severe diarrhea in suckling piglets was identified in Southern China in 2017. To develop an antigen that is specific, sensitive, and easy to prepare for serological diagnosis, antigenic sites in the SADS-CoV nucleocapsid (N) protein were screened. We generated and characterized an N-reactive monoclonal antibody (mAb) 3E9 from mice immunized with recombinant N protein. Through fine epitope mapping of mAb 3E9 using a panel of eukaryotic-expressed polypeptides with GFP-tags, we identified the motif 343DAPVFTPAP351 as the minimal unit of the linear B-cell epitope recognized by mAb 3E9. Protein sequence alignment indicated that 343DAPVFTPAP351 was highly conserved in different SADS-CoV strains and SADS-related coronaviruses from bat, with one substitution in this motif in HKU2-related bat coronavirus. Using mAb 3E9, we observed that N protein was expressed in the cytoplasm and was in the nucleolus during SADS-CoV replication. N protein was immunoprecipitated from SADS-CoV-infected Vero E6 cells. Taken together, our results indicated that 3E9 mAb could be a useful tool to investigate the structure and function of N protein during viral replication.

The baculovirus ChaB proteins are conserved in all completely sequenced Lepidopteran NPVs and are annotated as putative DNA binding proteins. Here we investigated Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) ORF51 (ha51), one of the ChaB homologues in HearNPV. 5'-RACE revealed that Ha51 is transcribed from a conventional early promoter transcriptional initiator motif (CATT) located at 159nt upstream of ATG. RT-PCR confirmed that ha51 is an early transcribed gene. To study the function of Ha51 in the life cycle of HearNPV, Ha51 knockout and repair bacmids were generated by homologous recombination in Escherichia coli. Growth curve and DNA replication analyses showed that the levels of budded virus (BV) production and viral DNA accumulation were significantly higher in cells infected with Ha51 null virus than those infected with wild-type bacmid derived virus. Electron microscopy revealed that polyhedra formation was not affected by the deletion of Ha51. Bioassay demonstrated that the Ha51-deleted virus had similar oral infectivity as the wild-type and rescued virus. Western blot analyses suggested that HA51 is a component of the nucleocapsid of BV and occlusion-derived virus as well as the envelope of BV. Immunofluorescence microscopy showed that HA51 protein is mainly localized in the cytoplasm of infected cells. Taken together, our results indicate that, unlike previously characterized baculovirual ChaB genes, Ha51 is involved in viral DNA replication and BV production and is transcribed in the early stage of infection.

The mature HIV-1 nucleocapsid protein (NCp7) is generated by sequential proteolytic cleavage of precursor proteins containing additional C-terminal peptides: NCp15 (NCp7-spacer peptide 2 (SP2)-p6); and NCp9 (NCp7-SP2). Here, we compare the nucleic acid chaperone activities of the three proteins, using reconstituted systems that model the annealing and elongation steps in tRNA(Lys3)-primed (-) strong-stop DNA synthesis and subsequent minus-strand transfer. The maximum levels of annealing are similar for all of the proteins, but there are important differences in their ability to facilitate reverse transcriptase (RT)-catalyzed DNA extension. Thus, at low concentrations, NCp9 has the greatest activity, but with increasing concentrations, DNA synthesis is significantly reduced. This finding reflects NCp9's strong nucleic acid binding affinity (associated with the highly basic SP2 domain) as well as its slow dissociation kinetics, which together limit the ability of RT to traverse the nucleic acid template. NCp15 has the poorest activity of the three proteins due to its acidic p6 domain. Indeed, mutants with alanine substitutions for the acidic residues in p6 have improved chaperone function. Collectively, these data can be correlated with the known biological properties of NCp9 and NCp15 mutant virions and help to explain why mature NC has evolved as the critical cofactor for efficient virus replication and long-term viral fitness.