The following points highlight the three main steps involved in the process of reproduction in mammalian viruses. The steps are: 1. Attachment 2. Entry into the Host Cell and Release of the Nucleic Acid 3. Biosynthesis of Viral Nucleic Acids and Proteins.
Step # 1. Attachment:
Attachment involves binding of the virion to the plasma membrane of the host cell. This binding takes place at specific sites of the host cell membrane where specific receptor proteins are located. Similarly, the virion — whether enveloped or naked — carries specific sites for attachment. Attachment occurs usually by random collision of the virion with the attachment site of the host.
The receptor is very often a glycoprotein which functions under normal conditions in uptake of essential molecules, or sometimes as immunoglobulin’s. For example, the receptor protein of influenza virus is a sialic acid containing protein, the receptor of human immunodeficiency virus (HIV) is CD4 protein, and that for poliovirus is 1CAM protein. In some cases, a co-receptor is involved in the binding process.
For example, in binding of HIV to the lymphocytes, a co-receptor called fusin is required in addition to the CD4 receptor protein. As all receptor proteins are not present in all types of cells of a particular host, specific viruses gain entry into the mammalian body by different routes.
The virions — whether enveloped or naked, have attachment sites on their surface. For example, the adenoviruses which have naked virions contain the penton fibers projecting from the corners of icosahedrons with which they get attached to the specific receptor sites of the host cells.
On the other hand, the enveloped virions of influenza virus (Orthomyxovirus) possess two kinds of surface projections (spikes or peplomers), one of which having haemagglutinin activity helps in attachment to the sialic acid residue of the host cell glycoprotein receptor.
Step # 2. Entry into the Host Cell and Release of the Nucleic Acid:
Soon after the virus particle is firmly attached to the cell membrane of a mammalian host, the virus enters into the cell. The mode of entry differs in different viruses. In rare cases, e.g. in poliovirus, the nucleic acid alone enters into the cell, while the naked capsid of these viruses is left outside the cell.
In this case, physical penetration of the intact virion into the host cell does not take place. A similar mode of entry is common among bacteriophages, but it is rare among mammalian viruses. In mammalian viruses, generally the entire virion enters into the host cell mainly in two ways. In one type of entry, the entire virion — whether naked or enveloped — is taken in by the host cell by a process of endocytosis.
The virion remains engulfed for the time being in an endocytic vesicle. This mode of entry is also known as viropexis. Another mode of entry operates only in some of enveloped viruses. It involves fusion of the viral envelope and the cell membrane of the host and results in release of the nucleocapsid in the cell cytoplasm e.g. the HIV.
The three modes of entry of mammalian viruses mentioned above are diagrammatically represented in Fig. 6.11:
A similar mode of entry is observed also in enveloped virions e.g. influenza virus. The intact virion with the envelope is internalized by endocytosis.
Except for the viruses which inject only the nucleic acid into the host cell — as the poliovirus — the nucleic acid of the virus has to be released from the nucleocapsid or the enveloped virion in the cytoplasm of the host cell. This process is known as un-coating and it differs from virus to virus.
Un-coating of nucleic acid is a poorly understood process. In case of poliovirus, un-coating seems to occur while the virus is still attached to the host cell membrane and results in entry of the nucleic acid into the host cell by an unknown mechanism. In case of other viruses, the virion physically enters into the host cell where the capsid is broken down to release the nucleic acid.
The viruses internalized by endocytosis are attacked by the host lysosomal enzymes to break down the capsid proteins. In case of complex viruses, like poxvirus, un-coating is a complicated process. Poxviruses enter the host cells by receptor-mediated endocytic pathway. The central core of the virion containing DNA and DNA- dependent RNA polymerase is released from the endocytic vesicles. The polymerase directs the synthesis of an enzyme protein that completes the un-coating process.
Step # 3. Biosynthesis of Viral Nucleic Acids and Proteins:
Upon un-coating, the viral nucleic acid is set free in the host cell. The events which follow differ in DNA and RNA viruses, and are also dependent on the strandedness of the nucleic acid, as also in case of single-stranded RNA viruses on the + and – nature of the nucleic acid.
The details of the biosynthetic process in DNA and RNA viruses are, therefore, discussed separately:
A. DNA Viruses:
Among the DNA viruses infecting mammals, only the Parvoviruses have a single-stranded genome and the others have double-stranded DNA (see Table 6.3). In the majority of DNA viruses, the uncoated viral DNA enters into the nucleus of the host cell where it is transcribed into viral messenger RNA molecules. Poxviruses are exceptions in this regard, because synthesis of all components occurs in the cytoplasm of the host cell.
In most of the viruses, the biosynthetic process proceeds in two phases, the early phase and the late phase. Generally, the early transcripts directing synthesis of early proteins are involved in replication of the viral nucleic acid, and the late transcripts produce enzymes required for synthesis of structural proteins of the virions. Both early and late proteins are synthesized in the cytoplasm of the host cell with the help of the biochemical machinery of the host cell.
Parvoviruses have a small single-stranded DNA genome of about 4.8 kb which permits synthesis of only three capsid polypeptides of the viruses. These viruses are, therefore, solely dependent on host enzymes for replication. The ss-DNA replicates in the nucleus, like most other DNA viruses.
The ss- DNA presumably functions as a template for synthesis of a complementary strand to yield a ds-DNA. It is not definitely known whether transcription occurs from the ss-DNA or ds-DNA. However, since the viral genome possesses no information for synthesis of other proteins, except the capsid proteins, the enzymes necessary for replication and transcription are obtained from the host cells.
Herpes viruses represent a group of enveloped ds-DNA viruses having a linear genome of about 160 kb. After un-coating, the DNA is transcribed by the host RNA polymerase and the transcripts are translated to form early proteins including the enzymes required for the replication of viral DNA.
The linear viral DNA then forms a circular molecule and replication of this DNA takes place by a viral DNA polymerase synthesized in the early phase. The late proteins are synthesized and the nucleocapsids of the progeny viruses are assembled.
As the progeny nucleocapsids are released from the nucleus where they are assembled, they acquire the envelope from the nuclear membrane of host cells. The envelope also contains some virus specific proteins, specially the glycoproteins.
Reproduction of Herpes-virus is schematically represented in Fig. 6.12:
Pox viruses, like varipla or vaccinia viruses, are the largest among mammalian viruses containing a ds-DNA within a complex virion. The virus enters by an endocytic pathway, but unlike other DNA viruses, their nucleic acid does not enter into the nucleus of the host cell.
The central core of the pox virion, released by un-coating contains in addition to the genome, an enzyme (An RNA polymerase) which synthesizes early m-RNAs. One of these direct synthesis of an enzyme which breaks down the nucleic acid core to release the viral DNA in the cytoplasm of the host cell. The replication of viral DNA, transcription of DNA and translation of viral proteins, all processes occur in the cytoplasm of the host cell. Assembly of new virions, too, takes place in the cytoplasm.
The Hepadna virus causing hepatitis B in man, have a circular ds-DNA genome. An exceptional feature is the presence of the enzyme reverse transcriptase in its virion, a characteristic shared only by the HIV which is an RNA virus. In Hepadnavirus, the viral DNA is formed from m-RNA by the reverse transcriptase.
After un-coating, the virion DNA is released in the nucleus of the host cell where it is transcribed into m-RNAs with the help of host cell enzyme. The m-RNAs are transported into the cytoplasm and translated. Transcription of viral DNA also produces a large RNA (about 3.4 kb) which forms a pre-genome. This RNA produces a DNA strand through the action of the reverse transcriptase. Subsequently, the DNA becomes double-stranded, is circularized and is packaged into the capsid to form virions.
The sequence of events in replication of Hepadnavirus is depicted below:
B. RNA Viruses:
Majority of RNA viruses have single-stranded RNA as genome. Only the reoviruses have a ds-RNA genome. The virions of Retroviruses have two identical single stranded RNA and they also carry two molecules of reverse transcriptase in the core of their virions.
The ds-RNA of Reoviruses is segmented in ten non-overlapping fragments. The naked icosahedral virion of Reovirus after un-coating releases the ds-RNA segments in the cytoplasm along with an RNA polymerase present in the core of the virion. The ds-RNA segments cannot function as m-RNA.
They are first transcribed by the viral RNA polymerase present in the virion to produce several m-RNAs. One or more of these m-RNAs direct the synthesis of a new RNA polymerase which transcribes the parental RNA segments. Of the ten segments of parental ds-RNA segments, three appear to be involved in the synthesis of core proteins and the rest in the synthesis of capside proteins.
The two strands of ds-RNA are designated as + or sense strand and as – or antisense strand. The – strand of ds-RNA is transcribed by RNA polymerase to produce a + strand which acts as m-RNA.
The events in replication of Reoviruses are represented next:
The Picornaviruses have naked icosahedral virions (21-30 nm) with single-stranded RNA (+) genome, causing poliomyelitis and common cold. The poliovirus attaches to the specific lipoprotein receptors on the host cell membrane and the capsids are structurally altered to liberate the viral RNA (+) into the cell cytoplasm. The RNA can function directly as m-RNA.
Initially, the (+) strand of the RNA directs the synthesis of an RNA-dependent RNA polymerase (replicase) which forms a (-) strand RNA to generate a replicative intermediate. This intermediate is a partially double- stranded RNA consisting of one complete strand and several growing complementary strands. From the replicative intermediate single-stranded (+) RNA molecules are formed which are incorporated into the progeny virions.
The virion RNA, on the other hand, which is a long polycistronic m-RNA produces by translation a long polypeptide which is cleaved into four different capsid proteins and enzymes. Assembly of progeny virions takes place in the cytoplasm of the host cell by packaging (+) RNA strand into the capsids.
The events in replication of Picornaviruses are shown below:
The Orthomyxo-viruses (influenza virus) and Paramyxoviruses (mumps and measles viruses) are enveloped, with (-) single-strand RNA genomes. The influenza virus attaches with its spikes to host cell receptors and enters into the cell by the endocytic pathway. Un-coating of the envelope in the endocytic vesicle releases the nucleocapsid into the cytoplasm of the host cell.
The nucleoprotein core then enters into the nucleus through nuclear membrane pore. Once inside the nucleus, the viral ss-RNA (-) begins to replicate. The (-) strands are copied into (+) strands by an RNA-dependent RNA polymerase (replicase) which is of viral origin.
The (+) RNA strands are transported into the cytoplasm where they function as m-RNAs and are translated into viral proteins. It should be noted that most of the RNA viruses replicate in the cytoplasm, but influenza viruses are exceptions in this regard, because they enter into the nucleus for replication.
Some of (+) strands generated by the replicase action remain in the nucleus and produce complementary (-) strands which are eventually used for packaging into the progeny virions. It is also to be noted that the virion (-) ss RNA of influenza virus is segmented and each segment forms a separate (+) RNA which acts as m-RNA. The spike proteins of influenza virus viz. haemagglutinin and neuraminidase and the virion matrix protein are synthesized in the cytoplasm along with the capsid proteins.
The spike and matrix proteins are then inserted into the cytoplasmic membrane of the host cell. As the completed nucleoprotein core of the influenza virus leaves the host cell by budding, it acquires the envelope from the cell membrane.
The events in replication of influenza virus are shown next and in Fig. 6.13:
Unique among the RNA viruses are the Retroviruses which include among others the human immunodeficiency virus (HIV) causing acquired immunodeficiency syndrome (AIDS). Though Retroviruses have single-stranded (+) RNA genome, their mode of replication differs from other (+) RNA viruses in that they produce DNA from RNA with the help of a virus-borne reverse transcriptase (RT) and in that the DNA is integrated into the host DNA.
HIV virion contains two identical RNA molecules and two molecules of reverse transcriptase in its nucleoprotein core. Reverse transcriptase is an RNA-dependent DNA polymerase. It copies the (+) RNA strand into a complementary (-) DNA single strand to form a (+) RNA / (-) DNA heteroduplex. Then an RNAse, called ribonuclease H, which is a component of the reverse transcriptase, degrades the (+) RNA strand. The reverse transcriptase then copies the (-) DNA strand to produce a (+) DNA strand and the two strands from a double helix.
HIV attaches to host cells (T-lymphocytes) containing CD4 receptor proteins on their membrane and enters by the endocytic pathway. Un-coating releases the viral RNA molecules and the reverse transcriptase. The RNA molecules are used as template for synthesis of double-stranded DNA.
The DNA molecule circularizes is transported into the nucleus and become integrated in one of the host chromosomes, where it is called a provirus. As provirus, it can continue indefinitely as part of the host chromosome. When activated, the ds-DNA of the provirus transcribes viral m-RNA with the help of host RNA polymerase. These messengers are then translated to capsid and envelope proteins, as well as reverse transcriptase.
The viral DNA also forms (+) RNA strand for incorporation into the progeny virions. The progeny virions escape by budding and on their way out they acquire the envelope from the cell membrane. The viral envelope proteins, already synthesized are incorporated into the host membrane system.
The sequence of events in the replication of the retrovirus HIV is shown in Fig. 6.14: 