2.5.1Hepatitis C Virus Description and Structural Proteins
HCV is an enveloped, single-stranded positive-sense RNA virus (Penin, 2003). Due to its considerable sequence heterogeneity HCV is classified as a separate genus in the Flaviviridae family and distinguished into six major genotypes showing a fairly different geographic distribution (Cocquerel et al., 1998). Its genome consists of 5‟ and 3‟ non coding regions and a single open reading frame that encodes a single viral polyprotein of 3010-3033 amino acids (Cocquerel, 2000; Chung and Wasan, 2004). The viral polyprotein undergoes post-translational cleavages to form functional viral proteins, both structural (core and envelope proteins) and non-structural (NS2-NS5 proteins), which produce the enzymes required for viral growth and replication (Cocquerel, 2000; Chung and Wasan, 2004). Because of its rapid replication and the high rate of error insertion of the RNA-dependent RNA polymerase, HCV spontaneously mutates within a given infected individual, resulting in related but distinct “quasispecies (Coito et al., 2004). The generation of these mutants appears to be one of the key mechanisms by which HCV escape the host‟s immune response, maintaining persistent infection (Cormier et al., 2004).
Very importantly, the replication cycle of the HCV occurs totally in the cytoplasm and - once the replication is stopped - the virus can be cleared from the cells and thus the infection definitively cured.
2.5.2Anatomy of Hepatitis C Virus
The structure of the hepatitis C virus is like that of most complex viruses – a core of genetic material (RNA), surrounded by a protective shell of protein, and further encased in a lipid (fatty) envelope of cellular material. However, the fact that the genetic information of the virus is stored in RNA, not DNA, has important consequences in the life cycle of the virus, and gives hepatitis C its dangerous ability to mutate (Everett, 2015).
All organisms, with the exception of the RNA viruses, store their permanent information in DNA, using RNA only as a temporary messenger for information. DNA is quite a stable molecule, not particularly reactive with other molecules, and the processes which
24
reproduce it make very few mistakes in the process of copying the molecule (between one in 1 million and 1 in 10 million). Most of these mistakes are normally corrected even when they do occur. This makes DNA an ideal format for the storage of information, for mutations (errors) only rarely occur, and most are not significant (Everett, 2015).
RNA, by contrast, is a quite reactive molecule, capable of reacting even with itself under the correct conditions. It also makes frequent mistakes during copying - averaging one mistake per 10,000 nucleotides each time it is copied. These properties make RNA verypoorly suited for the storage of information.
However, these very properties make RNA ideal for the storage of viral information.
Once the immune system has learned to recognize an infecting virus and create antibodies against it (developed immunity), it can quickly destroy it, so the virus can no longer use that host for reproduction. In order to re-infect a host - it must first change its nature enough that the immune system will no longer recognize it - in other words, it must mutate (Everett, 2015).Hepatitis C, as an RNA virus, has a powerful reproductive strategy. Because it stores its information in a "sense" strand of RNA, the viral RNA itself can be directly read by the host cell's ribosomes, functioning like the normal mRNA present in the cell. The virus thus needs no special abilities of its own - it uses the cell's own ribosomes to produce everything it needs for its takeover of the cell's processes and reproduction. This means hepatitis C requires only a small amount of RNA to encode its core information, and thus has lots of room for genetic variation within the non-essential portions of its RNA. This also gives it fewer common characteristics that can be readily identified by the immune system - or, for that matter, exploited by scientists working to create a treatment (Everett, 2015). Figure 2.5.1a below showed the HCV structure by electron microscopy.
25
Plate 2.5a: Hepatitis C virus Structure by Electron Microscopy(Everett, 2015)
26 2.5.3. Brief Life Cycle of Hepatitis C Virus
The life cycle of hepatitis C virus (HCV) has been briefly summarized here in six consecutive steps:(a) Virus binding and internalization (b) cytoplasmic release and uncoating (c) IRES-mediated translation and polyprotein processing (d) RNA replication (e) packaging and assembly (f) virion maturation and release. The topology of HCV structural and nonstructural proteins at the endoplasmic reticulum membrane is shown schematically (Bartenschlager et al., 2004). HCV RNA replication occurs in a specific membrane alteration, the membranous web. Note that IRES-mediated translation and polyprotein processing as well as membranous web formation and RNA replication, illustrated here as separate steps for simplicity, may occur in a tightly coupled fashion (Moradpour, 2007).
2.5.4 Characteristics and Functions of HCV Proteins
The HCV open reading frames (ORF) contain 9024 to 9111 nucleotides depending on the genotype. The ORF encodes at least 11 proteins, including 3 structural proteins (C or core, E1 and E2), a small protein, p7, whose function has not yet been definitively defined, 6 nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B), and the so-called
“F” protein which results from a frameshift in the core coding region. Table 2.5 shows HCV proteins and their roles in the viral lifecycle (Bartenschlager et al., 2004).HCV has evolved various strategies to counteract the host immune response and to establish persistent infection. Previous work has identified the HCV NS3-4A serine protease as a key viral protein blocking innate immune sensing pathways. NS3-4A cleaves and inactivates the essential adaptor molecule MAVS in the RIG-I viral RNA-sensing pathway, thereby blocking interferon production. More recently, Bartenschlager and his co-researchers have pursued a quantitative proteomics-based approach to identify novel cellular targets of the HCV NS3-4A protease. These studies revealed novel host targets that are currently being characterized for their role in the viral life cycle as well as in the pathogenesis of hepatitis C.
27
Figure 2.5b: Hepatitis C Virus Life Cycle(Moradpour, 2007)
28
Table 2.5: HCV proteins and their functions in the viral life cycle(Bartenschlager et al., 2004)
HCV protein Function
Apparent molecular weight (kDa)
Core Nucleocapsid 23(precursor)
21 (mature)
F/ARFaprotein Not yet known 16–17
E1 Envelope
Fusion domain?
33–35
E2 Envelope
Receptor binding Fusion domain?
70–72
p7 Calcium ion channel (viroporin) 7
NS2 NS2-3 autoprotease 21–23
NS3 Component of NS2-3 and NS3-4A proteinases
NTPase/helicase
69
NS4A NS3-4A proteinase cofactor 6
NS4B Membranous web induction 27
NS5A RNA replication by formation of replication complexes
56 (basal form)
58 (hyper-phosphorylated form)
NS5B RNA-dependent RNA polymerase 68
a. Frame shift /alternate reading frame
29
2.6 Hepatitis B and C Viruses Virologic Interaction
HCV “core” protein strongly inhibits HBV replication as early „90s in vitro studies showed (Schüttler et al., 2002; Chen et al., 2003). Two subsequent reports indicated that also the HCV NS5A protein may influence HBV activity, although they produced contrasting data in terms of inhibition or enhancement of the HBV replication (Guo et al., 2007; Pan et al., 2007). However, when the in vitro co-transfection experiments were conducted with full-length HBV genomes and HCV replicons (thus, not limiting the study to a single HCV protein) it was shown that the two viruses could replicate in the same hepatocyte without evidence of interference (Eyre et al., 2009), and that hepatocytes with replicating HBV could be infected by HCV without super-infection exclusion (Bellecave et al., 2009).Rodríguez-Iñigo and his co-researchers reported the possible co-existence of HBV and HCV in the same hepatocytes form liver biopsy specimens (Rodríguez-Iñigo et al., 2005). Because of several limitations, however, the transformed-hepatocyte cell culture systems used so far are not ideal for exploring the co-existence of the two viruses and, consequently, the experimental data available at present do not definitively clarify the possible interaction between them. Similarly, an in vivo model to study the dynamic process of a possible reciprocal interference in the replicative cycle and the production of respective viral proteins is not yet available (Zoulim, 2013).
The HBV and HCV virological patterns have also been investigated in quite a large number of clinical studies. Most of these studies were cross-sectional evaluation of the viral load of the two viruses at a single time point, showing an apparent dominant role of the HCV (high HCV RNA and low HBV DNA levels) in the majority of the cases. Other reports, however, suggest a reciprocal interference or even a dominant effect of HBV and ethnic factors have also been proposed to influence the dominant role of one virus on the other (Nguyen et al., 2011). In the middle of the last decade, an Italian multicenter study longitudinally examined a large series of HBsAg and anti-HCV antibody positive patients and showed that a wide and complex spectrum of virological profiles may occur in cases of co-infection. In fact, about one third of the cases presented broad changes over time of the amount of circulating HBV DNA or - less frequently - HCV RNA, thus revealing alternate phases of activity of one or both viruses. In this context, one should consider that the typical anti-HBe positive chronic hepatitis B is often characterized by phases of low levels of HBV replication interspersed with episodes of viral reactivation (Raimondo et al.,
30
1990), and many HBsAg/anti-HCV cases are anti-HBe positive. Similarly, also HCV - although infrequently - may show alternating phases of active and suppressed replication also in cases of single infection (Arase et al., 2000). In the context of the hypothesized interaction between the two viruses and particularly of the inhibitory effect operated by HCV on HBV, some anecdotal reports concerning co-infected patients treated with interferon (IFN) therapy for the productive HCV infection had showed the reactivation of the previously, apparently suppressed HBV once a favorable response to therapy had been achieved as shown by the permanent disappearance of serum HCV RNA (Chuang et al., 2006). Therefore, curing the HCV infection would produce the loss of the suppression on HBV that may reactivate. However, a more recent study longitudinally evaluating the behavior of apparently inactive HBV infection in patients under treatment for the simultaneous HCV infection showed that the inactive HBV status was maintained independently of the HCV response to therapy in all but two non-responder cases with persistently high HCV viremia levels who showed HBV DNA flares during the antiviral treatment, thus indicating a status of productive HBV infection with fluctuating virological profiles and suggesting that the HBV activity can be independent of the HCV during anti-HCV therapy (Saitta et al., 2006).
The immunology of the HBV-HCV dual infection has been evaluated in a few studies focused on T-lymphocyte response and analyzing small numbers of cases (Urbani et al., 2005). Larger studies are needed to better clarify the complex immunological aspects of this condition.
2.7 Human Immunodeficiency Virus
Human immunodeficiency virus (HIV) was discovered by the Pasteur National Institute of Health in 1983 as having a similar structure to the lymphadenopathy-associated virus (LAV) which was discovered few months earlier in immunocompromised patients (Knipe, 2001). The virus was classified as a member of the lentivirus genus and named HIV-1.
Later in 1986, another strain of the HIV was discovered on the West coast of Africa (Knipe, 2001). HIV-2 is distinct from HIV-1 in that it is less pathogenic and bears extra viral proteins than HIV-1. Though HIV-1 and HIV-2 have almost the same set of genes and very similar pathological effects, HIV-2 bears greater resemblance to the Simian immunodeficiency virus (SIV) (Pooper et al., 1999).HIV-1 exhibits all the structural characteristics of a typical lentivirus retroviridae. It was originally named a retrovirus
31
because of its particle associated reverse transcriptase, a hallmark of the retrovirus family.
It is a 100-120µL envelope virion with two single RNA strands and its similarity to morphology of the lentivirus genus confirmed its retrovirus status. HIV-1 uses the host cell membrane to form viral envelope. This envelope is covered by gp 41 and gp 120 surface and major histocompatibility complex class II (MHC-II) proteins inserted into the lipid envelope. Inside the lipid envelope the matrix formed gag protein 17 that holds the RNA-containing core in place. The cylindrical core not only stores the viral RNA and various proteins, it also contains complimentary RNA synthesized by the viral reverse transcriptase (Mandell et al., 2004).