Does Nitric Oxide Play a Critical Role in Viral Infections? (1998)

Many virus infections elicit vigorous host immune responses, both innate and acquired. The immune responses are frequently successful in controlling and then clearing the virus, using both cellular effectors such as natural killer (NK) cells and cytolytic T lymphocytes and soluble factors such as interferons (IFNs). However, some immune responses lead to pathologic changes or are unable to prevent the pathogen’s growth. This review will not be devoted to the different strategies viruses have taken to promote their transmission or survival but rather to one aspect of the innate immune response to infection: the role of nitric oxide (NO) in the antiviral repertoire. Recently, data from many laboratories, using both RNA and DNA viruses in experimental systems, have implicated a role for NO in the immune response. The data do not indicate a magic bullet for all systems but suggest that NO may inhibit an early stage in viral replication and thus prevent viral spread, promoting viral clearance and recovery of the host. 

The earliest host responses to viral infections are nonspecific and involve the induction of cytokines, among them, IFNs and tumor necrosis factor alpha (TNF-α). Gamma IFN (IFN-γ) and TNF-α have both been shown to be active in many cell types and induce cascades of downstream mediators (reviewed in references 25, 34, and 41). Others have found that NO synthase type 2 (NOS-2, iNOS) is an IFN-γ-inducible protein in macrophages, requiring IRF-1 as a transcription factor (12, 17). We have observed that the isoform expressed in neurons, NOS-1, is IFN-γ, TNF-α, and interleukin-12 (IL-12) inducible (20). Thus, NOS falls into the category of IFN-inducible proteins, activated during innate immune responses.

NO is produced by the enzymatic modification of l-arginine to l-citrulline and requires many cofactors, including tetrahydrobiopterine, calmodulin, NADPH, and O2. NO rapidly reacts with proteins or with H2O2 to form ONOO−, peroxynitrite, which is highly toxic (Fig. ​(Fig.1).1). NO also readily binds heme proteins, including Hb and its own enzyme. 



FIG. 1

Reaction of NO with proteins or H2O2 to form ONOO−.



This review will not include a great deal of detail about the biochemistry, pharmacology, and molecular biology of NOSs as there are many excellent review articles available (7, 10, 31, 41). However, to show the range of processes in which NOSs are involved, we will illustrate a few. NO was initially described as a physiological mediator of endothelial cell relaxation, an important role in hypotension (35, 42). An extension of this role is in penile erection (8). NO is central to long-term potentiation in neurons (32) and to activity in the biological clock, the suprachiasmic nucleus (13). There are three well-characterized isoforms of NOS, termed NOS-1, NOS-2, and NOS-3 (Table ​(Table11).



TABLE 1

Isoforms of NOS 



Immunologically, NOS activity, NOS-immunoreactive proteins, and mRNA have been found in autoimmune diseases, such as multiple sclerosis, associated with demyelinating lesions (11) and arthritic joints (40) and are thought to contribute to disease pathogenesis. NOS is frequently observed to be induced during the immune response (5). In contrast, in many intracellular bacterial and parasitic infectious diseases, NOS activity has been observed to be essential in eliminating pathogens such as Plasmodium falciparum (4).

In the last 5 years, dozens of articles have been published which show some association between NO and viral infections both in vivo and in vitro. There have been three basic experimental strategies used to determine if NO functionally inhibits viral replication: (i) using NO donors such as sodium nitroprusside (SNP), S-nitroso-l-acetylpenicillamine (SNAP), or 3-morpholino-sydononimine (SIN-1) in vitro; (ii) using analogs of the substrate l-arginine, such as l-NMA, l-NAME, 7-nitroindazole, to inhibit enzyme activity in vitro or in vivo; (iii) infecting host strains of mice which are homozygously deficient (knockouts) in one of the NOS isoforms. Table ​Table22 summarizes many of the findings in RNA and DNA viral systems. 



TABLE 2

Summary of published findings on the effect(s) of NO on viral infection 



In vitro, for most (but not all) viruses studied, prior activation of the cell to have enzyme activity before infection is associated with inhibition of viral replication. This has been accomplished by providing NO donors, by coculture with activated macrophages as a source of diffusing NO, or by directly activating NOS in cells with cytokines or through other cell surface receptors (e.g., the glutamate receptor, NMDA-R). This includes both DNA and RNA viruses, enveloped and encapsidated: all picornaviruses tested (20, 27, 28, 33, 39, 45), Japanese encephalitis virus (JEV) (26), mouse hepatitis virus (MHV) (24), vesicular stomatitis virus (VSV) (20), Friend murine leukemia virus (MuLV) (3), herpes simplex virus type 1 (HSV-1) (18, 20), vaccinia virus (16, 18, 30), and ectromelia virus (18).

There are several exceptions, some tested with positive controls of IFN-mediated viral inhibition (41), including influenza virus (20), Sindbis virus (20, 44), and tick-borne encephalitis virus (23) (note that another member of the Flaviviridae, JEV, is sensitive [26]). Thus, NO is not a magic bullet in vitro; however, it is very potent for many different viruses.

The mechanism of inhibition of viral replication in vitro is actively under investigation in many laboratories. It may be that there will be several different pathways involved, especially given the diversity of virus families which are sensitive or resistant. For coxsackievirus type B3 (CVB3) and JEV, RNA synthesis and protein synthesis are inhibited (26, 45). For VSV, very early protein synthesis is inhibited and the viral structural proteins are nitrosylated (40a). For vaccinia virus, late viral protein synthesis and DNA replication are inhibited (16, 30).

In some cases, NOS-2 is detectable in tissues from infected animals and may be attributable to activation of macrophages and microglia by IFN-γ or TNF-α. This has been associated with tissue pathology in several systems: CVB3 (28), borna virus (2, 22), MHV demyelination (14), human immunodeficiency virus (HIV) gp120 neuropathology (36), and HSV-1 pneumonia (1).

In vivo, the data on inhibition of viral replication tend to agree with the in vitro findings. That is, when a virus is very sensitive to NO in tissue culture, treatment of infected hosts with an inhibitor of the enzyme is associated with increased viral replication. This was found for VSV (20), Friend MuLV (3), HSV-1 (1), and ectromelia virus (18). An exception was observed with Sindbis virus, since two laboratories found that there was no in vitro sensitivity to NO (20, 44) but mice treated with an inhibitor succumbed more readily during central nervous system (CNS) infection (44); however, this drug was provided in drinking water, which may not have been palatable to sick mice. Exceptions to the correlation were observed for vaccinia virus and for MHV infection of the CNS, for which there was no effect on disease when mice were treated with an inhibitor of NOS (24, 37). In the murine cytomegalovirus (MCMV) system, treatment of mice with l-NMA suggested that NO played a more important role in controlling viral replication in the livers of mice, but NK cells were essential in the spleen for eliminating MCMV (43).

Although there are knockout mice for each of the three isoforms of NOS, the experiments with two of the systems have not yet been published. We have found that CNS infection of mice with VSV, which replicates in neurons, requires NOS-1 for clearance of infection, recovery, and survival (20a). NOS-3-deficient mice resembled wild-type mice in their responses to the infection (20a). Lowenstein’s work with NOS-2-deficient mice indicates that CVB3 replicates to higher titers in knockout mice (28a).

What does this all mean? NO frequently is an important mediator in intracellular inhibition of viral replication, which results in lower viral yields and more efficient host clearance of the infection, hence recovery. NO is not the only intracellular inhibitor, because many of the IFN-inducible proteins block viral pathways (41). There is no clear-cut way of predicting if NO will have a role in viral clearance or pathogenesis. DNA and RNA viruses are both sensitive or resistant.

There are many pathogens which are not inhibited by NO; however, NO may also contribute to tissue damage, especially if substantial numbers of macrophages are activated, producing large quantities of NO, as in Borna disease (2, 22) or HSV-1 pneumonia (1). Since there are many enzyme inhibitors available, those diseases in which NOS-2 activity is detrimental may benefit from enzyme antagonism. Host organ tropism also does not predict the selectivity of this response. However, in the case of viral encephalitis due to infection with picornaviruses, rhabdoviruses, HSV-1, or JEV, for instance, activation of NOS-1 may be lifesaving.