Signals at the bacteriophage phi 29 DNA replication origins required for protein p6 binding and activity.

Protein p6 of Bacillus subtilis phage phi 29 binds specifically to the ends of the viral DNA that contain the replication origins, giving rise to a nucleoprotein structure. DNA regions recognized by protein p6 have been mapped by deletion analysis and DNase I footprinting. Main protein p6-recognition signals have been located between nucleotides 62 and 125 at the right phi 29 DNA end and between nucleotides 46 and 68 at the left end. In addition, recognition signals are also present at other sites within 200-300 bp at each phi 29 DNA end. Protein p6 does not seem to recognize a specific sequence in the DNA, but rather a structural feature, which could be bendability. The formation of the protein p6-DNA nucleoprotein complex is likely to be the structural basis for the protein p6 activity in the initiation of replication.     

Functions of the HIV-1 matrix protein p17

HIV-1 replication is a dynamic process influenced by a combination of viral and host factors. The HIV-1 matrix protein p17 is a structural protein critically involved in most stages of the life cycle of the retrovirus. It participates in the early stages of virus replication as well as in RNA targeting to the plasma membrane, incorporation of the envelope into virions and particle assembly. Besides its well established functions, p17 acts as a viral cytokine that works on preactivated--but not on resting--human T cells promoting proliferation, proinflammatory cytokines release and HIV-1 replication after binding to a cellular receptor (p17R). Thus, p17 might play a key role in the complex network of host- and virus-derived stimulatory factors contributing to create a favourable environment for HIV-1 infection and replication. Here, we present a brief overview of the functions played by the matrix protein p17 in the HIV-1 life cycle and summarize the current understanding of how p17 could contribute to the pathogenesis of HIV-1 disease.     

In vivo assembly of phage phi 29 replication protein p1 into membrane-associated multimeric structures

The mechanisms underlying compartmentalization of prokaryotic DNA replication are largely unknown. In the case of the Bacillus subtilis phage 29, the viral protein p1 enhances the rate of in vivo viral DNA replication. Previous work showed that p1 generates highly ordered structures in vitro. We now show that protein p1, like integral membrane proteins, has an amphiphilic nature. Furthermore, immunoelectron microscopy studies reveal that p1 has a peripheral subcellular location. By combining in vivo chemical cross-linking and cell fractionation techniques, we also demonstrate that p1 assembles in infected cells into multimeric structures that are associated with the bacterial membrane. These structures exist both during viral DNA replication and when 29 DNA synthesis is blocked due to the lack of viral replisome components. In addition, protein p1 encoded by plasmid generates membrane-associated multimers and supports DNA replication of a p1-lacking mutant phage, suggesting that the pre-assembled structures are functional. We propose that a phage structure assembled on the cell membrane provides a specific site for 29 DNA replication.     

Dynamic relocalization of phage phi 29 DNA during replication and the role of the viral protein p16.7

We have examined the localization of DNA replication of the Bacillus subtilis phage phi 29 by immunofluorescence. To determine where phage replication was localized within infected cells, we examined the distribution of phage replication proteins and the sites of incorporation of nucleotide analogues into phage DNA. On initiation of replication, the phage DNA localized to a single focus within the cell, nearly always towards one end of the host cell nucleoid. At later stages of the infection cycle, phage replication was found to have redistributed to multiple sites around the periphery of the nucleoid, just under the cell membrane. Towards the end of the cycle, phage DNA was once again redistributed to become located within the bulk of the nucleoid. Efficient redistribution of replicating phage DNA from the initial replication site to various sites surrounding the nucleoid was found to be dependent on the phage protein p16.7.     

Hepatitis C virus p7 protein is crucial for assembly and release of infectious virions

Hepatitis C virus (HCV) infection is associated with chronic liver disease and currently affects about 3% of the world population. Although much has been learned about the function of individual viral proteins, the role of the HCV p7 protein in virus replication is not known. Recent data, however, suggest that it forms ion channels that may be targeted by antiviral compounds. Moreover, this protein was shown to be essential for infectivity in chimpanzee. Employing the novel HCV infection system and using a genetic approach to investigate the function of p7 in the viral replication cycle, we find that this protein is essential for efficient assembly and release of infectious virions across divergent virus strains. We show that p7 promotes virus particle production in a genotype-specific manner most likely due to interactions with other viral factors. Virus entry, on the other hand, is largely independent of p7, as the specific infectivity of released virions with a defect in p7 was not affected. Together, these observations indicate that p7 is primarily involved in the late phase of the HCV replication cycle. Finally, we note that p7 variants from different isolates deviate substantially in their capacity to promote virus production, suggesting that p7 is an important virulence factor that may modulate fitness and in turn virus persistence and pathogenesis.     

The nonstructural p17 protein of a fusogenic bat-borne reovirus regulates viral replication in virus species- and host-specific manners

Nelson Bay orthoreovirus (NBV), a member of the family Reoviridae, genus Orthoreovirus, is a bat-borne virus that causes respiratory diseases in humans. NBV encodes two unique nonstructural proteins, fusion-associated small transmembrane (FAST) protein and p17 protein, in the S1 gene segment. FAST induces cell–cell fusion between infected cells and neighboring cells and the fusogenic activity is required for efficient viral replication. However, the function of p17 in the virus cycle is not fully understood. Here, various p17 mutant viruses including p17-deficient viruses were generated by a reverse genetics system for NBV. The results demonstrated that p17 is not essential for viral replication and does not play an important role in viral pathogenesis. On the other hand, NBV p17 regulated viral replication in a bat cell line but not in other human and animal cell lines. Nuclear localization of p17 is associated with the regulation of NBV replication in bat cells. We also found that p17 dramatically enhances the cell–cell fusion activity of NBV FAST protein for efficient replication in bat cells. Furthermore, we found that a protein homologue of NBV p17 from another bat-borne orthoreovirus, but not those of avian orthoreovirus or baboon orthoreovirus, also supported efficient viral replication in bat cells using a p17-deficient virus-based complementation approach. These results provide critical insights into the functioning of the unique replication machinery of bat-borne viruses in their natural hosts.     

Deletions at the N terminus of bacteriophage phi 29 protein p6: DNA binding and activity in phi 29 DNA replication

Deletions corresponding to the first 5 or 13 amino acids (aa), not counting the initial Met, have been introduced into the N terminus of the phage phi 29 protein p6. The activity of such proteins in the in vitro phi 29 DNA replication system, their capacity to interact with the phi 29 DNA ends, and their interference with the wild type (wt) protein p6 activity have been studied. The initiation activity of protein p6 decreased considerably when 5 as were deleted and was undetectable when 13 aa were removed. The mutant proteins were unable to specifically interact with the phi 29 DNA ends. These results indicate the need of an intact N terminus for the activity of protein p6. However, such N-truncated proteins inhibited both the specific binding of the wt protein p6 to the phi 29 DNA ends and its activity in phi 29 DNA replication.     

The phage phi29 membrane protein p16.7, involved in DNA replication, is required for efficient ejection of the viral genome

It is becoming clear that in vivo phage DNA ejection is not a mere passive process. In most cases, both phage and host proteins seem to be involved in pulling at least part of the viral DNA inside the cell. The DNA ejection mechanism of Bacillus subtilis bacteriophage phi29 is a two-step process where the linear DNA penetrates the cell with a right-left polarity. In the first step approximately 65% of the DNA is pushed into the cell. In the second step, the remaining DNA is actively pulled into the cytoplasm. This step requires protein p17, which is encoded by the right-side early operon that is ejected during the first push step. The membrane protein p16.7, also encoded by the right-side early operon, is known to play an important role in membrane-associated phage DNA replication. In this work we show that, in addition, p16.7 is required for efficient execution of the second pull step of DNA ejection.     

Characterization of the bacteriophage phi29-encoded protein p16.7: a membrane protein involved in phage DNA replication

An early expressed operon, located at the right end of the linear bacteriophage phi29 genome, contains open reading frame (ORF)16.7, whose deduced protein sequence of 130 amino acids is conserved in phi29-related phages. Here, we show that this ORF actually encodes a protein, p16.7, which is abundantly and early expressed after infection. p16.7 is a membrane protein, and the N-terminally located transmembrane-spanning domain is required for its membrane localization. The variant p16.7A, in which the N-terminal membrane anchor was replaced by a histidine-tag, was purified and characterized. Purified p16.7A was shown to form dimers in solution. To study the in vivo role of p16.7, a phi29 mutant containing a suppressible mutation in gene 16.7 was constructed. In vivo phage DNA replication was affected in the absence of p16.7, especially at early infection times. Based on the results, the putative role of p16.7 in in vivo phi29 DNA replication is discussed.     

Initiation of phage phi 29 DNA replication by the terminal protein modified at the carboxyl end

A mutant at the carboxyl end of the terminal protein, p3, of phage phi 29 DNA has been constructed by inserting an containing the stop translation codon TGA in the three possible reading frames, immediately downstream of a phage phi 29 DNA fragment coding for all but the last five amino acids of protein p3. The activity in the formation of the p3-dAMP initiation complex in vitro of this mutant as well as another one previously isolated, also mutated at the carboxyl end, have been tested. The results obtained suggest that an intact carboxyl end in the phage phi 29 terminal protein is essential for its normal primer function in DNA replication.     

Characterization of the phage phi 29 protein p5 as a single-stranded DNA binding protein. Function in phi 29 DNA-protein p3 replication.

The phage phi 29 protein p5, required in vivo in the elongation step of phi 29 DNA replication, was highly purified from Escherichia coli cells harbouring a gene 5-containing plasmid and from phi 29-infected Bacillus subtilis. The protein was characterized as the gene 5 product by amino acid analysis and NH2-terminal sequence determination. The purified protein p5 was shown to bind to single-stranded DNA and to protect it against nuclease degradation. No effect of protein p5 was observed either on the formation of the p3-dAMP initiation complex or on the rate of elongation. However, protein p5 greatly stimulated phi 29 DNA-protein p3 replication at incubation times where the replication in the absence of p5 leveled off.     

Assembly of phage phi 29 genome with viral protein p6 into a compact complex.

The formation of a multimeric nucleoprotein complex by the phage phi 29 dsDNA binding protein p6 at the phi 29 DNA replication origins, leads to activation of viral DNA replication. In the present study, we have analysed protein p6-DNA complexes formed in vitro along the 19.3 kb phi 29 genome by electron microscopy and micrococcal nuclease digestion, and estimated binding parameters. Under conditions that greatly favour protein-DNA interaction, the saturated phi 29 DNA-protein p6 complex appears as a rigid, rod-like, homogeneous structure. Complex formation was analysed also by a psoralen crosslinking procedure that did not disrupt complexes. The whole phi 29 genome appears, under saturating conditions, as an irregularly spaced array of complexes approximately 200-300 bp long; however, the size of these complexes varies from approximately 2 kb to 130 bp. The minimal size of the complexes, confirmed by micrococcal nuclease digestion, probably reflects a structural requirement for stability. The values obtained for the affinity constant (K(eff) approximately 10(5) M-1) and the cooperativity parameter (omega approximately 100) indicate that the complex is highly dynamic. These results, together with the high abundance of protein p6 in infected cells, lead us to propose that protein p6-DNA complexes could have, at least at some stages, during infection, a structural role in the organization of the phi 29 genome into a nucleoid-type, compact nucleoprotein complex.     

Viral stress-inducible protein p56 inhibits translation by blocking the interaction of eIF3 with the ternary complex eIF2.GTP.Met-tRNAi

Viral stress-inducible protein p56 is produced in response to viral stress-inducing agents such as double-stranded RNA and interferon, as well as other poorly understood mechanisms of viral infection. It has been shown previously that p56 is able to bind the eukaryotic initiation factor 3e(eIF3e) (p48/Int-6) subunit of the eukaryotic translation initiation factor eIF3 and function as an inhibitor of translation in vitro and in vivo. The exact mechanism by which p56 is able to interfere with protein synthesis is not understood. Based on the known roles of eIF3 in the initiation pathway, we employed assays designed to individually look at specific functions of eIF3 and the effect of p56 on these eIF3-mediated functions. These assays examined the effect of p56 on ribosome dissociation, the eIF3.eIF4F interaction, and enhancement of the ternary complex eIF2.GTP.Met-tRNAi formation. Here we report that p56 is able to inhibit translation initiation specifically at the level of eIF3.ternary complex formation. The effect of p56-mediated inhibition was also examined in two different contexts, cap-mediated and encephalomyocarditis virus internal ribosomal entry site-mediated translation. Whereas cap-dependent initiation was severely inhibited by p56, internal ribosomal entry site-mediated translation appeared to be insensitive to p56.