Direct penetration virus

One direction of research that could hasten the arrival of entry inhibitors to the clinics is the development of compounds that interact with conserved intermediates of the entry process or with the protein structures that, on binding to receptors, trigger the conformational changes that lead to the formation of these entry intermediates.

Typically, such intermediates are only transiently exposed, so viruses might not have evolved strategies to avoid inhibitors targeted to these structures. In addition, conserved intermediates are usually important for virus entry and presumably cannot easily be substituted by other structures after mutation. An example of the successful design of an entry inhibitor that shows proof of the concept is the 5-Helix protein, which interferes with a conserved intermediate in the entry of HIV-1 Ref.

A related example is a class of peptides that could have broad applications to several viruses containing class I fusion proteins. The peptides are derived from regions of fusion proteins heptad repeats that have a propensity to form coiled-coils and which serve as fusion intermediates and enable oligomerization of proteins. T also known as DP is derived from the carboxy-terminal heptad region of the HIV-1 gp41 and showed potent inhibitory activity in vivo Bossart and C.

Broder, personal communication , perhaps owing to the endocytic entry route of these viruses. Although peptides have certain promising features, including a relatively small size that might ensure good penetration combined with high binding affinity, the lack of oral formulations, short half-life, possible toxicity and immunogenicity might limit their application. Recently, a small molecule, BMS, was identified that inhibits the entry of a broad range of HIV-1 isolates by a mechanism which was attributed to competition with the CD4 receptor for binding to gp Ref.

So, at inhibitory concentrations, BMS does not interfere with CD4 binding, indicating a different inhibitory mechanism. By analogy with inhibitors of picornavirus entry, such as pleconaril, BMS might inhibit the entry of HIV-1 by binding to conserved structures that are important for the conformational changes which gp must undergo for viral entry Potent virus-specific inhibitors of the viral-membrane-merging step have not been identified yet.

Another promising direction is the development of multivalent inhibitors that can overcome problems caused by mutation of viral proteins to escape inhibition because multivalent inhibitors bind to several regions of the same or different protein s on the viral surface. One example of a multivalent inhibitor is the multimeric soluble receptors of influenza and HIV-1 Ref.

The use of entry inhibitors in combination or as fusion proteins could also result in increased efficiency. Finally, improvement of current methods for structure-based design by accounting for protein flexibility and dynamics in binding to ligands 85 , and screening methods for inhibitors 86 , 87 would certainly expand the range of possible inhibitors that can be tested.

Neutralizing antibodies and vaccine immunogen design. Neutralizing antibodies usually inhibit virus entry by preventing attachment of the virus to the cell or by binding to entry intermediates 88 , 89 , Human immunoglobulin composed of concentrated antibodies collected from pooled human plasma has been successfully used as a preventative treatment for virus infections, including rabies, hepatitis A and B, measles, mumps, varicella, cytomegalovirus and arenaviruses.

Antibodies can completely prevent infection, but once infection is established they are a much less efficient treatment. The only monoclonal antibody in clinical use today to treat a viral disease — Synagis MEDI — is more potent than the polyclonal immunoglobulin that is presently in use, and is broadly active against numerous RSV type A and B clinical isolates It binds to the F protein of RSV with high affinity 3 nM and inhibits virus entry and cell fusion in vitro with an IC 50 of approximately 0.

It seems that the efficacy of Synagis in vivo is correlated with the high affinity of binding and potency of this antibody in vitro The X-ray crystal structures of rhinovirus 21 and poliovirus 22 indicate a possible mechanism by which picornaviruses can avoid neutralization by antibodies through the mutation of non-conserved amino acid residues surrounding the receptor-binding site — a 2 nm deep and 2 nm wide canyon Fig.

It was initially hypothesized that the conserved amino acid residues of the canyon are not accessible by antibodies; however, it was later shown that a strongly neutralizing antibody, Fab17, can penetrate deep within the receptor-binding canyon by undergoing a large conformational change without inducing conformational changes in the virus 90 , Unusually, not only the hypervariable residues but also residues from the framework region of Fab17 contact the canyon.

Yet another remarkable mechanism of immune recognition of viruses is the recently discovered receptor mimicry by post-translational modification tyrosine sulphation of antibodies Ref. It seems that any accessible viral surface can be recognized by antibodies.

Rapidly mutating viruses can escape neutralizing antibodies even if they bind to structures that are essential for virus replication, such as receptor-binding sites, unless they bind with energetically identical profiles Whether a virus will escape neutralization by antibodies depends on the interplay between the antibody affinity avidity and kinetics of binding, generation rate, concentration and the viral mutation rate and fitness.

Mutations of immunodominant structural loops that form antibody-binding sites and mutations leading to changes in oligosaccharide attachment to viral entry proteins are common mechanisms by which viruses avoid neutralization 90 , Mutations of conserved residues that have a role in the entry mechanism typically result in reduction or loss of infectivity.

Antibodies or their derivatives that bind to epitopes where residues contribute most of the binding energy could have potential as entry inhibitors.

Epitopes that are exposed after virus binding to receptors are typically well conserved — for example, the 17b 39 and X5 Ref. One potential problem with using antibodies as entry inhibitors in this case could be limited access to the post-receptor-binding state of the viral entry protein due to the relatively large size of the whole antibody. Solving this, and other problems, could lead to the development of potent broadly neutralizing antibodies which could limit the generation of resistant viruses, especially if these inhibitors are used in combination with other antibodies or inhibitory molecules.

Many viruses, especially RNA viruses such as HIV-1, exist as swarms of virions inside an infected individual, and might significantly differ in sequence between isolates. So, elicitation of potent, broadly neutralizing antibodies is an important goal for vaccine development. However, elicitation of these antibodies in vivo has not been successful. Identification of broadly neutralizing antibodies and the characterization of their epitopes could help to design vaccine immunogens that would be able to elicit these neutralizing antibodies in vivo — so-called retrovaccinology At present, all vaccines that elicit antibodies against entry proteins have been developed empirically using an antigen, rather than by designing an immunogen on the basis of the antibodies produced.

The important advantages of human antibodies as therapeutics are low or negligible toxicity combined with high potency and a long half-life. However, drawbacks include the generation of neutralization-resistant virus mutants, limited access of the large antibody molecules to the site of virus replication, lack of oral formulations and the high cost of production and storage. Viruses are usually associated with disease. However, some viruses can be beneficial. The HERV-W Env, known as syncytin, is fusogenic and has a role in human trophoblast cell fusion and differentiation Retroviral particles have been observed in the placenta, along with fused placental cells, which are morphologically reminiscent of virally induced syncytia.

These studies led to the proposal that an ancient retroviral infection might have been a pivotal event in mammalian evolution Viruses have long been used to transfer genes into cells.

During the last decade, another important application has been the viral delivery of genes and drugs to treat cancer. A major challenge has been to develop virus entry proteins to deliver molecules to specific cells with high efficiency.

To achieve this goal it is often desirable to engineer viruses that do not infect cells expressing the native receptor, but instead target a cell of choice. Engineering of entry proteins in this way is known as transductional retargeting A conceptually simple approach to transductional retargeting is to incorporate the protein that determines cell tropism into the infecting virion of choice — known as virus 'pseudotyping'.

This has been used in both retroviruses and adenoviruses, and does not require prior knowledge of specific virus—receptor interactions. In a related approach, viral entry proteins are used to produce drug and gene delivery vehicles, for example, the F protein of Sendai virus has been incorporated into liposomes to form virosomes and the L protein of hepatitis B has been incorporated into yeast-derived lipid vesicles Retargeting of retroviruses, adenoviruses and AAVs has been achieved by conjugation of entry proteins with molecular adaptors, such as bi-specific antibodies that have particular receptor-binding properties.

Modification of the entry proteins so that the normal receptor-binding property is abolished, or a ligand for alternative receptor binding is incorporated has also been successful at redirecting adenovirus tropism in cell culture, but is unlikely to work for the entry of viruses that require receptor-induced conformational changes, such as retroviruses, unless detailed molecular mechanisms of those conformational changes are better understood.

A related approach is based on screening libraries of chimaeric Envs from different strains of MLV , or randomized peptides inserted at tolerant sites in viral proteins, such as VP3 of AAV This approach seems promising for the selection of specific retargeting vectors.

Understanding the structure of AAV and other viruses could help to further improve the specificity and efficiency of retargeting. Retargeting viruses with complex entry mechanisms that involve several proteins, such as those of herpes viruses and poxviruses, remains challenging. Elucidation of the molecular mechanisms and the dynamics of the conformational changes driving virus entry remains a significant challenge.

It requires the development of new approaches to study the rapid conformational changes of a small number of membrane-interacting protein molecules that are surrounded by many more non-interacting molecules. A more realistic goal is the determination of the structures of proteins that mediate the entry of all human viruses and the identification of the cognate cellular receptors.

If research continues at the present pace, this goal could be accomplished within the next decade. Identification of all the cellular receptors for human viruses would be an important contribution to our understanding of virus tropism and pathogenesis. The various, and in many cases unexpected, ways that entry proteins can affect pathogenesis could offer new opportunities for intervention. The development of panels of human monoclonal antibodies against every entry-related protein of all pathogenic human viruses could accelerate our understanding of entry mechanisms and help to fight viral diseases.

Recent progress in virus retargeting also raises hopes for the possibility of designing entry machines that can deliver genes and other molecules to any cell of choice. Google Scholar. Sieczkarski, S. Dissecting virus entry via endocytosis. Pelkmans, L. Local actin polymerization and dynamin recruitment in SVinduced internalization of caveolae.

Science , — Dimitrov, D. Cell biology of virus entry. Cell , — Rawat, S. Modulation of entry of enveloped viruses by cholesterol and sphingolipids. Takeda, M. Influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion. Natl Acad. USA , — Waarts, B. Lack of correlation with lipid raft formation in target liposomes. Kielian, M. Specific roles for lipids in virus fusion and exit. Igakura, T.

Bomsel, M. Entry of viruses through the epithelial barrier: pathogenic trickery. Nature Rev. Cell Biol. Seisenberger, G. Real-time single-molecule imaging of the infection pathway of an adeno-associated virus. Lowy, R. Observation of single influenza virus-cell fusion and measurement by fluorescence video microscopy. USA 87 , — Lakadamyali, M. Visualizing infection of individual influenza viruses. Virology , — Quantitation of HIV-1 infection kinetics.

White, J. Fusion of Semliki forest virus with the plasma membrane can be induced by low pH. Shows that a low pH can trigger rapid and efficient fusion of SFV with plasma membranes, which leads to delivery of the viral genome in a form that is suitable for replication. Carr, C. Influenza hemagglutinin is spring-loaded by a metastable native conformation. USA 94 , — Proposes that the native structure of HA is trapped in a metastable state and that the fusogenic conformation is released by destabilization of the native structure.

Hogle, J. Poliovirus cell entry: common structural themes in viral cell entry pathways. Stubbs, M. Anthrax X-rayed: new opportunities for biodefence. Trends Pharmacol. Chen, Y. SNARE-mediated membrane fusion.

Rossmann, M. Structure of a human common cold virus and functional relationship to other picornaviruses. Nature , — Three-dimensional structure of poliovirus at 2. References 21 and 22 describe the first crystal structures of human viruses with important implications for understanding their mechanisms of entry and design of inhibitors.

Mancini, E. Cryo-electron microscopy reveals the functional organization of an enveloped virus, Semliki Forest virus. Cell 5 , — Kuhn, R.

Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Chappell, J. EMBO J. Structural analysis reveals evolutionary relationships between two unrelated virus families. Wilson, I. The first, and still the most informative structure of an entry envelope glycoprotein — the influenza HA. Chen, L. The structure of the fusion glycoprotein of Newcastle disease virus suggests a novel paradigm for the molecular mechanism of membrane fusion. Structure 9 , — Rey, F.

The first structure of a class II fusion protein that reveals an entirely unexpected configuration that is dramatically different from the structure of class I fusion proteins. Modis, Y. A ligand-binding pocket in the dengue virus envelope glycoprotein. Lescar, J. The fusion glycoprotein shell of Semliki Forest virus: an icosahedral assembly primed for fusogenic activation at endosomal pH.

The second structure of a class II fusion protein, which enabled the classification of these proteins as distinct from class I. Heinz, F. The machinery for flavivirus fusion with host cell membranes. Colman, P. The structural biology of type I viral membrane fusion.

The assay plates are incubated and specimens showing no plaques on any of the assay plates pass the test, while plates that exhibit plaque-forming units fail. Generic filters. Viral Penetration Test. Online Form. Non-enveloped viruses can enter the cytosol by directly penetrating the plasma membrane, as well as through a variety of endocytic mechanisms leading to penetration of internal membrane s. Internal membranes crossed by non-enveloped viruses include the endosomal membrane e.

SV40; Pelkmans et al. Strategies to disrupt or traverse host cell membranes must be included in the mechanisms of non-enveloped virus entry. However, the precise molecular and biophysical means by which non-enveloped viruses gain entry to the cytosol have not been clearly defined in all cases. Early electron micrographs of PV-infected cells demonstrated virus particles adjacent to and directly penetrating the plasma membrane Dunnebacke et al. Such evidence gave rise to a long-standing belief that PV entry occurs directly at the cell surface.

However, use of drugs such as the ionophore monensin to dissipate cellular proton gradients suggested a pH-dependent route of PV entry Madshus et al. It was known that under low pH, substances such as diphtheria toxin exposed a hydrophobic region which could insert into the membrane Sandvig and Olsnes These data suggest PV association with cellular receptors induced the exposure of hydrophobic regions at low pH.

Although it is possible that micro-regions of low pH occur at the cell surface, apart from specialised tissues such as the stomach, extracellular fluid is typically neutral, suggesting that PV enters cells via an acidic intracellular compartment. Recent developments allowing the imaging of fluorescent PV in live cells have supported an endocytic internalisation route Brandenburg et al.

Furthermore, the involvement of actin in the entry process was implicated by inhibition of infection following incubation with cytochalasin D. Use of siRNA and inhibitors of clathrin-, caveolin- and flotillin-dependent pathways as well as macropinocytosis suggest that PV entry is independent of all these cellular pathways Brandenburg et al.

This study also used total internal reflection fluorescence microscopy to demonstrate that genome release into the cytosol occurs from recently internalised vesicles, close to the cell surface. Taken together, recent studies of PV entry highlight an endocytic event that is independent of the classical clathrin- and caveolin-mediated pathways, followed by low pH-mediated exposure of hydrophobic residues in an early endocytic vesicle.

Recent reports of clathrin- and caveolin-independent routes of endocytosis await further clarification reviewed in Kirkham and Parton , and it is likely that studies of PV entry will provide important information about alternative endocytic mechanisms operating in host cells. Although SV40 represents a virus with a particularly well-studied entry pathway, recent evidence suggests that this well-characterised route is not as clear cut as it may initially have seemed.

SV40 became of particular interest to virologists and cell biologists alike as being the first virus shown to enter cells via a clathrin-independent endocytic pathway Anderson et al. In fact it was, at least in part, the study of SV40 entry which led to the discovery of what we know as caveolar endocytosis and the caveosome, and as such, labelled SV40 is often used as a tool to study intracellular trafficking Fig.

Caveolar endocytosis of SV Early studies on SV40 entry found the internalisation pathway to differ in several ways from the classical clathrin-mediated endocytic process described for many viruses. Firstly, electron micrographs of SVinfected cells showed the majority of particles in small tight-fitting vesicles, which appeared to be uncoated Hummeler et al.

These vesicles were able to fuse to generate larger compartments within the cell which we now term caveosomes; Maul et al. Moreover, the ability of SV40 to internalise into different endocytic compartments was dependent on the capacity to be enveloped by cellular membranes.

Subsequently, the caveolar entry pathway of SV40 was confirmed by the use of drugs found to disrupt caveolae, such as nystatin, which inhibited SV40 entry Anderson et al. However, as relatively stable membrane domains, it was unclear how caveolae could offer a productive entry pathway for SV Furthermore, murine polyoma virus binding to host cells induces an up-regulation of primary and early response genes that regulate particle internalisation Zullo et al.

SV40—host cell interactions induce an intracellular signalling event that up-regulates the expression of primary response genes c-myc, c-jun, c-cis within 30 min and JE, a PDGF-inducible gene encoding monocyte chemoattractant MCP-1, within 90 min Dangoria et al. SV40 up-regulation of both c-myc and c-jun was blocked by the tyrosine kinase inhibitor genistein.

Furthermore, genistein was shown to block SV40 infection in a reversible manner, resulting in a model where SV40 binding induces a signalling pathway that primes particle internalisation. More recently, labelling of the virus and use of live-cell imaging have demonstrated a role for actin in SV40 internalisation and trafficking Pelkmans et al. Upon virus binding, a transient breakdown of actin-stress fibres was observed.

After leaving the caveosome, labelled SV40 trafficked in tubular, caveolin-free vesicles, which move along microtubules to fuse with smooth ER organelles Pelkmans et al. Recent data showing SV40 entry into cells lacking detectable caveolae, including the human hepatoma Huh7 cell line and embryonic fibroblasts from a Cav1 knockout mouse, suggest alternative pathways may be in operation Damm et al.

The same authors noted caveolin-independent SV40 internalisation in wild-type embryonic fibroblasts with an active caveolar pathway Damm et al. In all of the aforementioned cells, viruses were seen to internalise in small, tight-fitting vesicles similar to those seen in the first EM images of SVinfected cells. SV40 was subsequently transported to pH-neutral organelles, which resembled caveosomes despite being devoid of both Cav1 and Cav2 Damm et al.

Importantly, expression of SV40 encoded T-antigen suggests these caveolin-independent pathway s represent productive infection. The observation that this entry mechanism was observed in cells with detectable Cav1, indicates that this alternative pathway is active even in the presence of Cav1. AdV 2 and 5 are non-oncogenic adenoviruses that infect the upper respiratory tract.

CAR is expressed at different levels on different tissues and is the primary determinant for susceptibility to Ad infection Bergelson et al. The first electron micrographs depicting AdV 2 entry suggested a role for coated vesicles early in the entry process Brown and Burlingham ; Svensson Furthermore, the inhibitory effects of lysosomotropic agents on infection indicated that this was followed by escape from acidic endosomes Svensson The use of drugs known to selectively inhibit receptor-mediated endocytosis reduced the extent of virus internalisation similarly to effects on transferrin entry a marker for clathrin-mediated endocytosis; Varga Despite the mounting evidence for a clathrin-mediated entry pathway Fig.

Clathrin-mediated endocytosis of Ad2. Electron microscopy of HeLa cells incubated with Ad2 demonstrates internalisation of virus through clathrin-coated pits into clathrin-coated vesicles adapted from Meier et al Although the use of dominant negative constructs of Eps15 required for clathrin-mediated endocytosis and dynamin required for vesicle fission seemed to confirm the clathrin-mediated entry pathway, AdV was reported to stimulate the uptake of fluid phase markers, largely via macropinocytosis Wang et al.

This is followed by trafficking to the early endosome, where low pH mediates endosomal escape Gastaldelli et al. Alongside endosomal escape, macropinocytosis is stimulated leading to the non-specific internalisation of virus particles from the extracellular environment. It is unclear whether this is a coincidental side effect of adenovirus infection, or whether this represents a manipulation of host cell machinery to ensure the uptake of more virions.

As CAR is a component of tight junctions, it is inaccessible to virus on the apical side of the epithelium, for example, viral particles in the intestinal lumen. Virus attachment to DAF on the apical surface is thought to trigger signalling events culminating in actin rearrangement and virus movement to the tight junction Coyne and Bergelson ; Coyne et al.

Signalling events induced by virus interaction with DAF are thought to induce caveolar endocytosis of virus at the tight junction Coyne et al. Moreover, although not involved in CVB binding, particle internalisation from the tight junction is dependent on occludin Coyne et al. Cell polarity and viral receptors localised to the tight junction are also important factors in hepatitis C virus entry, which will be discussed below.

Infection with an enveloped virus requires the fusion of the viral envelope with a cellular membrane. In some cases, this can occur at the plasma membrane, as reported for HIV, where binding to plasma membrane-expressed forms of CD4 and chemokine receptors induce changes in the viral envelope glycoprotein that are thought to mediate membrane fusion under neutral pH conditions. Fusion of other enveloped viruses occurs within the low-pH environment of an acidic endosomal compartment.

Enveloped viruses typically reach the endosomal compartment via trafficking in clathrin-coated vesicles, although a caveolar route of entry has been reported for human coronavirus E Nomura et al.

Examples of enveloped viruses with entry mechanisms of particular interest are HCV, influenza A and HIV and will be discussed in more detail below. Hepatitis C virus is a hepatotropic enveloped virus associated with liver disease, fibrosis, cirrhosis and hepatocellular carcinoma. The HCV particle comprises a single-stranded positive-sense RNA surrounded by an icosahedral capsid and envelope derived from a host cell lipid bilayer Bartosch and Cosset HCV encodes two envelope glycoproteins E1 and E2 which play a critical role in binding host cell surface receptors and membrane fusion Keck et al.

Following development of these experimental systems, the tight junction proteins Claudin-1 and Occludin were recently reported to be essential for virus internalisation Evans et al. HCVpp bearing diverse glycoproteins of all major genotypes show a marked preference for infecting liver-derived cells suggesting that receptor-dependent entry events may in part define hepatotropism Bartosch et al. Hepatocytes in the liver are highly polarised with bile canaliculi, surrounded by tight junctions, running between adjacent cells at the apical membrane.

HCV enters the liver via the sinusoidal blood and is likely to encounter the basolateral surface of hepatocytes.

The involvement of tight junction proteins has raised many questions about HCV entry. For example, does the virus need to locate to tight junctions to internalise? Current data demonstrate that HCV E2 engagement of CD81 promotes clathrin-mediated endocytosis and there is limited evidence to support a role for CDinduced movement of the virus to tight junctions in polarised hepatoma cells Farquhar, personal communication. Soon after the development of HCVpp and HCVcc, the use of drugs such as Bafilomycin A1 and concanamycin A which inhibit the vacuolar ATPase, dissipating membrane proton gradients demonstrated the pH-dependence of HCV entry, implicating the involvement of receptor-mediated endocytosis and fusion in an acidic endosomal compartment Hsu et al.

Furthermore, the use of dominant negative constructs of Eps15 and dynamin demonstrated a clathrin-mediated endocytic entry process Meertens et al. A recent siRNA study confirmed the involvement of several genes involved in clathrin-mediated endocytosis and actin polymerisation in the viral entry process and used time-lapse imaging to observe viral entry in live unpolarised liver-derived cells Coller et al.

The authors demonstrated an association between labelled HCV and clathrin, suggesting that virions bind to filopodia and traffic towards the cell body, where endocytosis occurs.

However, analysis of viral particles in polarised cells will be necessary before the exact entry pathway of the virus is clear. The entry process of Influenza A is a much-studied topic and differs from the simple membrane fusion processes described for the majority of enveloped viruses. A virus polymerase associated with the nucleocapsid, transcribes the virus RNA while the latter is still within the capsid. Many of the enveloped viruses and certain non-enveloped viruses enter the host cell through engulfment by receptor-mediated endocytosis and form coated vesicles.

The virions attach to coated pits with the protein clathrin and the pits then pinch off to form coated vesicles filled with viruses.

These vesicles fuse with lysosomes after the clathrin has been removed. Lysosomal enzymes help in un-coating of virion inside the cytoplasm. Un-coating is the process of separation of viral genome from the protein coat.

Though the process of un-coating is not fully understood, it is proclaimed that the lysosomal enzymes help in animal virus un-coating by degrading the capsid and low endosomal pHs often trigger the process of un-coating. It has been reported in some cases that the viral envelop fuses with the lysosomal membrane and the partially degraded capsid along with viral genome nucleocapsid is released into the host cytoplasm.

Once in the cytoplasm, viral genome may be released from the capsid upon completion of un-coating or may function while still attacked to capsid components. However, in some DNA viruses the replication takes place in cytoplasm e. Certain late genes direct the synthesis of capsid proteins.

The latter spontaneously self-assemble to form the capsid. It appears that in case of icosahedral viruses the capsid protein assembly first forms procapsid in which the viral genome is inserted by some unknown mechanism.

However, in case of enveloped viruses the capsid protein assembly is generally similar to that of naked viruses poxvirus is exception.

The capsids of these viruses are assembled in the cell cytoplasm by a lengthy, complex procedure that begins with the enclosure of a portion of cytoplasmic matrix through construction of a new membrane. Now the newly synthesized viral DNA condenses, passes through the membrane, and moves to the centre of the immature virus. Release of newly formed animal viruses from the host cell differs between naked and enveloped viruses.

The naked animal viruses are released most often by the lysis of the host cell. In enveloped viruses, however, the virus-encoded proteins are incorporated in the plasma membrane and then the nucleocapsid is simultaneously released; the envelop is formed by membrane-budding. Top Menu BiologyDiscussion. Bacteriophages: Classification and Morphological Groups.